Objective:To explore the effects of the immune responses mediated by topological structures of three-dimensional bioprinted scaffolds on hair follicle cycle in mice.Methods:The study was an experimental research. The alginate-gelatin composite hydrogels were printed into scaffolds using a three-dimensional bioprinter and named T45 scaffolds, T60 scaffolds, and T90 scaffolds according to the 3 topological structures of the scaffolds (the rotation angles of the printhead during printing were 45°, 60°, and 90°, respectively), and the morphology of the three scaffolds was observed after cross-linking by naked eyes. Nine 8-week-old female C57BL/6J mice were divided into T45 group, T60 group, and T90 group, according to the random number table, with three mice in each group, and the T45, T60, and T90 scaffolds were subcutaneously implanted on the back of mice, respectively. On post implantation day (PID) 7, the hair growth in the dorsal depilated area of mice was observed, the thickness of the fiber capsule around the scaffolds was observed by hematoxylin-eosin staining, and the expression levels of CD68, bone morphogenetic protein-2 (BMP-2), and tumor necrosis factor (TNF) protein in the tissue surrounding the scaffolds were observed by immunofluorescence staining. The samples of the above experiments were all 3.Results:The topological structures of the three scaffolds were all clear with high fidelity after cross-linking. On PID 7, the hair growth was obvious in the dorsal depilated area of mice in T45 group and T90 group, while hair growth was slow in the scaffold implantation area of mice in T60 group, which was significantly different from that of the unimplanted area. On PID 7, compared with (18±4) μm in T90 group, the thickness of both the fiber capsule around the scaffolds ((39±4) and (55±8) μm) of mice in T45 group and T60 group was significantly increased ( P<0.05); the thickness of the fiber capsule around the scaffolds of mice in T60 group was also significantly increased compared with that in T45 group ( P<0.05). On PID 7, the expression level of CD68 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05). The expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05), and the expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T45 group was significantly higher than that in T90 group ( P<0.05). The expression level of TNF protein in the tissue surrounding the scaffolds of mice in T60 group was significantly lower than the levels in T45 group and T90 group (with both P values <0.05). Conclusions:Three-dimensional bioprinted scaffolds with different topological structures mediate different degrees of immune responses after being implanted in mice. A moderate immune response promotes hair growth in depilated area of mice, while an excessive immune response results inhibits the hair follicle entering into the anagen phase.

Pulmonary fibrosis is the end-stage manifestation of a large class of lung diseases characterized by fibroblast proliferation and accumulation of a large amount of extracellular matrix accompanied by inflammatory injury and tissue structure destruction. Studies have shown that flavonoids have anti-pulmonary fibrosis effects through multiple paths， including dihydromyricetin， morin and fisetin can inhibit fibroblast differentiation； Trollius altaicus flavonoids， hesperidin and linarin can play an anti-inflammatory role； total flavonoids from Scutellaria baicalensis， scutellarin and chrysin can inhibit epithelial- mesenchymal transition； total flavonoids of Litchi chinensis， diosmin and silybin can play an anti-oxidative stress role； quercetin， baicalin and apigenin can regulate autophagy； total flavonoids of Oxytropis falcata， calycosin and dihydroquercetin can regulate apoptosis； naringin， scutellarin and total flavonoids of Dracocephalum moldavica can inhibit pyroptosis， thus exerting anti- pulmonary fibrosis effects.

OBJECTIVE To investigate the anti-pulmonary fibrosis effect of linarin in vivo and in vitro， and investigate its mechanism preliminarily. METHODS C57BL/6J mice were randomly divided into normal group （carboxymethylcellulose sodium）， model group （carboxymethylcellulose sodium）， positive control group （pirfenidone， 200 mg/kg）， linarin low-dose and high-dose groups （12.5， 25 mg/kg）， with 8 mice in each group. Except for normal group， pulmonary fibrosis model was induced in other groups. After modeling， they were given relevant medicine intragastrically， once a day， for consecutive 14 d. The general situation of mice was observed， and their lung indexes were measured； the levels of tumor necrosis factor-α （TNF-α） and transforming growth factor-β1（ TGF-β1） in serum and interleukin-6 （IL-6） in lung tissue were determined. Hematoxylin-eosin （HE） staining and Masson staining were used to observe the histopathological morphology of lung. The pulmonary fibrosis was scored according to Ashcroft score standard. The expressions of α-smooth muscle actin （α-SMA） and （type Ⅰ collagen， Collagen Ⅰ）， phosphorylated extracellular signal-regulated kinase （p-ERK1/2） and TGF-β1 in lung tissues were detected. HFL1 cells were stimulated by TGF- β1 to form pulmonary fibrosis model in vitro， which were divided into normal group， model group and linarin low-， medium- and high-concentration groups （3.7， 7.4， 14.8 mg/L）. After being cultured for 48 h， the protein expressions of α-SMA， Collagen Ⅰ and p-ERK1/2 in HFL1 cells were detected. RESULTS In vivo， compared with normal group， the lung index of model group and the levels of TNF- α， TGF- β1 and IL-6 were significantly increased （P＜0.01）. There were a large number of inflammatory infiltration and cellular fibrosis lesions in the alveoli， and a large number of collagen depositions. The scores of HE staining and Masson staining were significantly increased （P＜0.01）. The protein expressions of α-SMA， Collagen Ⅰ， p-ERK1/2 and TGF-β1 in lung tissue were up-regulated significantly （P＜0.01）. Compared with model group， above indexes of mice were improved significantly in linarin high-dose group （P＜0.05 or P＜0.01）， and most of indexes （except for lung index） were improved significantly in linarin low-dose group （P＜0.05 or P＜0.01）. In vitro， compared with blank group， the density of cells in the model group increased， and obvious proliferation and other changes occurred； protein expressions of α-SMA， Collagen Ⅰ and p-ERK1/2 were significantly up-regulated （P＜0.05 or P＜0.01）. Compared with model group， the cell density of each concentration group was decreased and the morphology gradually returned to normal； the expressions of above proteins in linarin high-concentration group and the protein expression of p-ERK1/2 in linarin medium-concentration group were down-regulated significantly（P＜0.05 or P＜0.01）. CONCLUSIONS Linarin may regulate ERK and inflammatory pathways to reduce the inflammatory response， thereby exerting anti-pulmonary fibrosis effect.

Renal interstitial fibrosis (RIF) is the crucial pathway in chronic kidney disease (CKD) leading to the end-stage renal failure. However, the underlying mechanism of Shen Qi Wan (SQW) on RIF is not fully understood. In the current study, we investigated the role of Aquaporin 1 (AQP1) in SQW on tubular epithelial-to-mesenchymal transition (EMT). A RIF mouse model induced by adenine and a TGF-β1-stimulated HK-2 cell model were etablished to explore the involvement of AQP 1 in the protective effect of SQW on EMT in vitro and in vivo. Subsequently, the molecular mechanism of SQW on EMT was explored in HK-2 cells with AQP1 knockdown. The results indicated that SQW alleviated kidney injury and renal collagen deposition in the kidneys of mice induced by adenine, increased the protein expression of E-cadherin and AQP1 expression, and decreased the expression of vimentin and α-smooth muscle actin (α-SMA). Similarly, treatmement with SQW-containing serum significantly halted EMT process in TGF-β1 stimulated HK-2 cells. The expression of snail and slug was significantly upregulated in HK-2 cells after knockdown of AQP1. AQP1 knockdown also increased the mRNA expression of vimentin and α-SMA, and decreased the expression of E-cadherin. The protein expression of vimentin increased, while the expression of E-cadherin and CK-18 significantly decreased after AQP1 knockdown in HK-2 cells. These results revealed that AQP1 knockdown promoted EMT. Furthermore, AQP1 knockdown abolished the protective effect of SQW-containing serum on EMT in HK-2 cells. In sum, SQW attentuates EMT process in RIF through upregulation of the expression of AQP1.
Drugs, Chinese Herbal/pharmacology* ; Humans ; Animals ; Mice ; Male ; Cell Line ; Rats ; Kidney/physiology* ; Fibrosis/drug therapy* ; Renal Insufficiency, Chronic/drug therapy* ; Adenine ; Epithelial-Mesenchymal Transition ; Aquaporin 1/metabolism*

Objective:To investigate the relationship between single nucleotide polymorphic (SNP) loci of PLCE1 gene and primary nephrotic syndrome (PNS) and its response to glucocorticoid therapy in Guangxi Zhuang children. Methods:It was a retrospective cohort study. One hundred and fifty-five Guangxi Zhuang children with PNS in the Affiliated Hospital of Youjiang Ethnic Medical College from October 2020 to May 2022, and 100 healthy Zhuang children during the same period as controls were included. Four SNP loci including rs17109674, rs10786156, rs3740360 and rs2274224 of PLCE1 gene were selected and high-throughput sequencing was used to analyze the genotypes. Logistic regression analysis model was used to analyze the correlation between each SNP locus and onset of PNS and steroid-resistant nephrotic syndrome. The SHEsis online software was used to analyze the link disequilibrium of each SNP locus, and construct the haploid type. Results:(1) Logistic regression analysis results showed that AC+CC genotype (AA as reference, OR=0.449, 95% CI 0.256-0.786, P=0.005), AC genotype (AA as reference, OR=0.354, 95% CI 0.188-0.667, P=0.001) and C allele gene (A as reference, OR=0.615, 95% CI 0.390-0.971, P=0.037) of rs3740360 were correlated with the risk of PNS in children. The genotypes and allele genes of rs17109674, rs10786156, rs3740360 and rs2274224 were not associated with the risk of steroid-resistant nephrotic syndrome in children (all P>0.05). (2) Strong linkage disequilibrium existed between rs10786156 and rs2274224 ( D'=0.702, r2=0.484). rs17109674 and rs10786156 ( D'=0.128, r2=0.007), rs17109674 and rs3740360 ( D'=0.142, r2=0.007), rs17109674 and rs2274224 ( D'=0.045, r2=0.001), rs10786156 and rs3740360 ( D'=0.255, r2=0.023), and rs3740360 and rs2274224 ( D'=0.281, r2=0.028) all had weak linkage disequilibrium. (3) The haploid AGCG ( OR=0.282, 95% CI 0.079-1.008, P=0.038), GGCC ( OR=0.327, 95% CI 0.111-0.967, P=0.034) and GGAG ( OR=4.616, 95% CI 1.179-18.069, P=0.016) were all correlated with the risk of PNS in children. Conclusions:AC genotype, AC+CC genotype, and C allele gene of rs3740360 SNP locus may reduce the risk of PNS in Guangxi Zhuang children. Haploid AGCG and GGCC may be associated with decreased incidence of PNS, while GGAG may be associated with increased incidence of PNS in Guangxi Zhuang children. The genotypes and alleles of 4 SNP loci are not associated with the risk of steroid-resistant nephrotic syndrome.

Objective:To analyze the mutation types and distribution characteristics of thalassemia gene among high-risk populations in Sanya City, and to evaluate the effectiveness of blood routine screening, in order to provide scientific basis for formulating measures for prevention and control of thalassemia in Sanya City.Methods:Retrospective analysis was used to collect detection results and clinical data from high-risk individuals who completed genetic screening for thalassemia at Sanya Materal and Child Health Hospital from January 2019 to August 2021. Mutation types and distribution characteristics of thalassemia gene were analyzed, and the missed detection rate and sensitivity of blood routine indicators [mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH)] were evaluated based on the results of genetic screening for thalassemia.Results:A total of 5 760 high-risk individuals were included in the screening results of thalassemia genes, and 3 868 samples of thalassemia gene mutations were detected, with a detection rate of 67.15%. Among them, there were 2 979 samples with α-thalassemia genetic mutations, with a detection rate of 51.72%; including 2 966 common genotype samples (99.56%), the main genotype was αα/-α 3.7 (20.14%, 600/2 979); 13 rare genotype samples (0.44%), 4 cases of αα/-- THAI, 3 cases of α CD40(AAG>AA-)α/αα, 2 cases of α PPα/αα, and 1 case of Fusion gene/αα, Fusion gene/α WSα, α WSα/α PPα, and α CD40(AAG>AA-)α/α WSα each. There were 340 samples with β-thalassemia gene mutations, with a detection rate of 5.90%; including 336 common genotype samples (98.82%). The β CD41/42/β N genotype was dominant (57.65%, 196/340); 4 rare genotype samples (1.18%), β CD5(-CT)/β N, β IVS-Ⅱ-2(-T)/β N, β IVS-Ⅱ-761(-T)/β N and β Initiation(ATG>AGG)/β N 1 case each. There were 549 samples of αβ-compound type thalassemia, with a detection rate of 9.53%. The α missing recombination β CD41/42 genotype was dominant (61.02%, 335/549). There were a total of 4 226 samples that could be traced back to MCV and MCH. Among them, 3 007 samples were found to have mutations in thalassemia genes through screening, 2 584 cases were found to have abnormalities in the combination of MCV and MCH indicators, and 423 samples were missed in blood routine screening, with a missed detection rate of 14.07% (423/3 007). The missed samples were mainly α static type, accounting for 89.13% (377/423) of the total missed samples. The screening sensitivity of MCV combined with MCH for α-, β- and αβ-compound type thalassemia was 82.65%, 98.07% and 98.15%, respectively. Conclusion:The types of genetic mutations in thalassemia in Sanya City are complex and diverse, and there are certain omissions in the blood routine screening of MCV combined with MCH.

Objective:To evaluate the prognostic value of pretreatment systemic immune-inflammation index (SII) and lactate dehydrogenase (LDH) in non-metastatic nasopharyngeal carcinoma (NPC).Methods:We retrospectively collected the data of 839 patients with non-metastatic NPC from National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital and Sun Yat-sen University Cancer Center between January 2007 and October 2015. All patients received intensity modulated radiation based treatment. Optimal cutoff value of SII and LDH were determined by X-title software. The association between SII, LDH and clinical prognosis of non-metastatic NPC patients were analyzed. Kaplan-Meier method was used for survival analysis, and Log rank test was used for comparison of survival rates between groups. Propensity score matching (PSM) analysis was carried out to minimize the effects of confounding factors. The risk stratification model of prognosis by combining N stage, SII and LDH was constructed to compare the prognosis of patients in high risk group, middle risk group and low risk group, and the receiver operating characteristic (ROC) curve analysis was used to evaluate its prognostic value.Results:The optimal cutoff value of SII is 447.2×10 9/L for predicting the 5-year overall survival (OS) of NPC patients, and the best cutoff value of LDH is 198.9 U/L. The proportion of patients with stage T3-4 and stage III-IVB in high SII group was higher than that in low SII group ( P<0.001). Multivariate Cox regression analysis showed that N stage, SII and LDH were independent factors of OS, progression-free survival (PFS) and distant metastasis-free survival (DMFS) of NPC patients (N stage, HR=1.705, 95% CI: 1.247-2.332; HR=1.755, 95% CI: 1.342-2.295; HR=2.161, 95% CI: 1.515-3.082. SII, HR=1.525, 95% CI: 1.097-2.119; HR=1.518, 95% CI: 1.150-2.004; HR=1.837, 95% CI: 1.272-2.653. LDH, HR=2.041, 95% CI: 1.403-2.968; HR=1.725, 95% CI: 1.233-2.414; HR=2.492, 95% CI: 1.690-3.672, respectively). After PSM, SII was still an independent prognostic factor of OS, PFS and DMFS in NPC patients ( HR=1.52, 95% CI: 1.09-2.12; HR=1.52, 95% CI: 1.15-2.00; HR=1.82, 95% CI: 1.26-2.63, respectively). Combined with N 2-3 stage, SII (>447.2×10 9/L), and LDH (>198.9 U/L), patients were divided into high-(3 risk factors), intermediate- (2 risk factors) and low-risk (0-1 risk factors) groups. The 5-year OS rates of patients in low-, intermediate- and high-risk groups were 86.1%, 79.8% and 41.2% respectively, the 5-year PFS rates were 80.7%, 70.2% and 33.9% respectively, and the 5-year DMFS rates were 88.9%, 79.2% and 47.5% respectively. There were significant differences in OS, PFS and DMFS among these three groups ( P<0.001). Distant metastasis was the main failure pattern in low-, intermediate- and high-risk groups, and the highest rate of distant metastasis was 83.3% (15/31) in high-risk group. ROC curve of the risk stratification model for predicting 5-year OS of NPC patients is 0.610, which is higher than TNM stage (0.609), SII (0.574) and LDH (0.558). Conclusions:Pretreatment SII and LDH are significantly correlated with the prognosis of patients with non-metastatic NPC. The combination of SII, LDH and N stage can stratify the prognostic risk of NPC patients. The risk stratification model can enhance the accuracy of prognosis.

Objective:To compare the specific IgE positive rates of the patients between allergic respiratory diseases and respiratory infectious diseases in Guangzhou, the relationship between the co-sensitization of house dust mite (HDM) allergen and Aspergillus fumigatus (AF) allergen and asthma, allergic rhinitis with asthma, pneumonia, upper respiratory infections, bronchitis, serum total immunoglobulin E (total Immunoglobulin E, tIgE) and age were analyzed, to provide the basis for the prevention and treatment of respiratory allergic diseases and respiratory infectious diseases in this area.Methods:A total of 2 535 patients with confirmed respiratory allergic diseases and respiratory infectious diseases were selected retrospectively from the outpatient or inpatient department of the First Affiliated Hospital of Guangzhou Medical University from April 2017 to June 2021 and detected HDM and AF specific IgE (sIgE) by the ImmunoCAP system. The age range was 1 to 89 years. The median age was 5 years. The average age was 9. ≤3 years old group n=894, 4-6 years old group n=721, 7-18 years old group n=615, 19-49 years old group n=207, >49 years old group n=98. There were 1 596 males (62.96%) and 939 females (37.04%). There were 1 279 cases of allergic diseases and 1 256 cases of respiratory infectious diseases. The different disease groups were divided into asthma group (411 cases), allergic rhinitis group (458 cases), allergic rhinitis combined with asthma group (410 cases), pneumonia group (463 cases), upper respiratory tract infection group (299 cases) and bronchitis group (494 cases). The difference of specific IgE (sIgE) and tIgE between HDM and AF was analyzed. For statistical analysis, continuous variables were tested by Mann-Whitney U. Classification data by chi-square test or Fisher′s exact test. Results:1 313 (51.79%) patients were sIgE positive for HDM allergen, 65 (2.56%) were sIgE positive for AF allergen, and 50 (1.97%) were both positive. In the respiratory allergic disease group, 877 cases (68.57%,877/1 279) were positive for HDM allergen sIgE, 57 cases (4.46%,57/1 279) were positive for AF allergen sIgE, and 44 cases (3.44%,44/1 279) were both positive; 436 cases (34.71%,436/1 256) of respiratory infectious diseases were positive for HDM allergen sIgE, 8 cases (0.64%,8/1 256) were positive for AF allergen sIgE, and 6 cases (0.48%,6/1 256) were both positive. In monosensitization, the HDM allergen sIgE sensitization rate was the highest in the allergic rhinitis & asthma group, at 80.24% (329/410). The positive rate of HDM allergen sIgE in male patients was 53.76%(858/1 596), and the positive rate in female patients was 46.22%(434/939), and the difference between the two was statistically significant (χ 2=13.449, P<0.001). In polysensitization, asthma patients (5.35%,22/411) had the highest positive rate of HDM sensitization with AF, followed by allergic rhinitis patients (3.06%,14/458), allergic rhinitis with asthma (1.95%,8/410). The positive rate of respiratory infectious diseases such as pneumonia (0.43%,2/463), upper respiratory infections (0.33%,1/299), and bronchitis (0.61%,3/494) with AF was extremely low. The positive rate of HDM combined with AF in infants(≤3 years old group,0.34%, 3/894; 4-6 years old group, 0.97%, 7/721)was significantly lower than that in teenagers and adults(7-18 years old group,3.58%, 22/615; 19-49 years old group,6.28%, 13/207;>49 years old group,5.10%, 5/98).In the patients with HDM and AF combined sensitization, HDM sIgE levels were distributed in all grades, and AF sIgE levels were mainly in grades 1, 2, and 3. Conclusion:The positive rate of HDM combined with AF was higher in patients with respiratory allergic diseases such as asthma, allergic rhinitis, and allergic rhinitis combined with asthma, suggesting that clinical attention should be paid to the combination of HDM and AF in patients with asthma and allergic rhinitis, especially adults, more likely to be combined with AF.

Objective:To investigate the printability and cytocompatibility of sodium alginate-gelatin (AG) bioink containing platelet-rich plasma derived from human umbilical cord blood (HUCB-PRP), named HUCB-PRP-AG bioink, and the effect of the three-dimensionally printed tissue with the bioink on full-thickness skin defect wounds in nude mice.Methods:The method of experimental research was used. HUCB-PRP-AG bioinks with 2.5%, 5.0%, and 10.0% of HUCB-PRP by volume were prepared and named 1P-AG, 2P-AG, and 4P-AG, respectively. The appearances of AG, 1P-AG, 2P-AG, and 4P-AG at room temperature were observed, and their viscosity and storage/loss modulus were measured by a rotational rheometer. The above four bioinks were used for three-dimensional bioprinting respectively, and the appearances of the printed tissue were observed (the printed tissue was subsequently cross-linked and used). The four kinds of bioprinted tissue were respectively co-cultured with human umbilical vein endothelial cells (HUVECs) in Transwell chambers with HUVEC special medium for 24 h, and the cell proliferation level was detected by cell counting kit 8 ( n=3). The four kinds of bioprinted tissue were respectively cultured in Dulbecco's modified eagle medium for 12, 24, and 48 h, which were dried and weighed, and the degradation rate was calculated ( n=3). The expression of vascular endothelial growth factor (VEGF) in the culture supernatant of 1P-AG, 2P-AG, or 4P-AG cultured in phosphate buffer solution at 0.5, 24.0, and 48.0 h was detected by enzyme-linked immunosorbent assay ( n=5). Sixteen female BALB/c-NU nude mice aged 6-8 weeks were selected to establish a full-thickness skin defect wound model on the back and were divided into conventional control group with wounds being covered with medical hydrocolloid dressing alone, HUCB-PRP group with additional HUCB-PRP dripping to the wounds, AG group additionally covered with AG printed tissue, and 4P-AG group additionally covered with 4P-AG printed tissue, respectively (with 4 nude mice in each group). The wound healing of 3 nude mice in each group was observed on post injury day (PID) 4, 8, and 14, and the wound healing rate was calculated. The wound tissue of the remaining nude mouse in each group was collected on PID 8, the histopathological changes were observed after hematoxylin and eosin staining, and the CD31-positive new blood vessels were observed after immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, least significant difference test, and Bonferroni correction. Results:At room temperature, AG, 1P-AG, 2P-AG, and 4P-AG were semi-transparent liquid, and AG was light yellow, while 1P-AG, 2P-AG, and 4P-AG were light red but the color successively deepened. The viscosity of AG, 1P-AG, 2P-AG, and 4P-AG decreased with the increase of shear rate at the temperature of 10 ℃ and shear rate of 0.1-10.0 s -1; the storage moduli of the four bioinks were greater than the loss moduli at the temperature of 10 ℃ and angular frequency range of 1-100 rad/s. Both the resolution and morphology of the printed tissue of four bioinks were similar. The proliferation levels of HUVECs co-cultured with 1P-AG, 2P-AG, and 4P-AG printed tissue for 24 h were 0.885±0.030, 1.126±0.032, and 1.156±0.045, respectively, which were significantly higher than 0.712±0.019 of HUVECs co-cultured with AG printed tissue ( P<0.01). The proliferation levels of HUVECs co-cultured with 2P-AG and 4P-AG printed tissue for 24 h were significantly higher than the level of HUVECs co-cultured with 1P-AG printed tissue ( P<0.01). The degradation rates of 1P-AG, 2P-AG, and 4P-AG printed tissue were significantly higher than those of AG printed tissue at 12, 24, and 48 h of culture ( P<0.01). The degradation rates of 2P-AG and 4P-AG printed tissue at 24 and 48 h of culture were significantly higher than those of 1P-AG printed tissue ( P<0.01). The degradation rate of 4P-AG printed tissue at 12 h of culture was significantly higher than that of 1P-AG printed tissue ( P<0.01), and the degradation rates of 4P-AG printed tissue at 24 and 48 h of culture were significantly higher than those of 2P-AG printed tissue ( P<0.01). At 0.5, 24.0, and 48.0 h of culture, the expressions of VEGF in the culture supernatant of 2P-AG printed tissue were significantly higher than those of 1P-AG printed tissue ( P<0.01), and the expressions of VEGF in the culture supernatant of 1P-AG and 2P-AG printed tissue were significantly lower than those of 4P-AG printed tissue ( P<0.01). The wounds of nude mice in conventional control group and HUCB-PRP group were dry and smaller on PID 8 compared with those on PID 4, and the wounds of nude mice in HUCB-PRP group were smaller with no scabs on PID 14 compared with those in conventional control group. The printed tissue on the wound of nude mice in AG and 4P-AG groups was significantly degraded with no obvious exudation being observed on the wounds on PID 4, the wounds were significantly epithelialized and smaller on PID 8, and there was no scab on the wound on PID 14. The wounds of nude mice in 4P-AG group were completely epithelialized on PID 14. Compared with those in conventional control group, the wound healing rate of nude mice in AG group was significantly decreased on PID 4 ( P<0.05), and the wound healing rates of nude mice in HUCB-PRP group and 4P-AG group at all time points after injury and in AG group on PID 8 and 14 were significantly increased ( P<0.01). Compared with those in HUCB-PRP group, the wound healing rates of nude mice were significantly decreased on PID 4 and 8 in AG group and on PID 4 in 4P-AG group ( P<0.01), while the wound healing rates of nude mice were significantly increased on PID 14 in AG group and on PID 8 and 14 in 4P-AG group ( P<0.01). The wound healing rate of nude mice in 4P-AG group was significantly higher than that in AG group at all time points after injury ( P<0.01). On PID 8, a large number of inflammatory cells infiltration, a small amount of new microvessels, and a small amount of CD31-positive new blood vessels were observed in the wounds of nude mice in conventional control group; a large number of inflammatory cells infiltration, abundant new microvessels, and quite a lot CD31-positive new blood vessels were observed in the wounds of nude mice in HUCB-PRP group; light inflammatory inflammation, a small amount of new microvessels, and a small amount of CD31-positive new blood vessels were observed in the wounds of nude mice in AG group; light inflammatory inflammation, a large number of new microvessels, and a large number of CD31-positive new blood vessels were observed in the wounds of nude mice in 4P-AG group. Conclusions:HUCB-PRP-AG bioink has good printability and cytocompatibility, and its three-dimensionally printed tissue can promote vascularization of full-thickness skin defect wounds in nude mice and accelerate wound healing.

Objective:To evaluate the prognostic value of pretreatment systemic immune-inflammation index (SII) and lactate dehydrogenase (LDH) in non-metastatic nasopharyngeal carcinoma (NPC).Methods:We retrospectively collected the data of 839 patients with non-metastatic NPC from National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital and Sun Yat-sen University Cancer Center between January 2007 and October 2015. All patients received intensity modulated radiation based treatment. Optimal cutoff value of SII and LDH were determined by X-title software. The association between SII, LDH and clinical prognosis of non-metastatic NPC patients were analyzed. Kaplan-Meier method was used for survival analysis, and Log rank test was used for comparison of survival rates between groups. Propensity score matching (PSM) analysis was carried out to minimize the effects of confounding factors. The risk stratification model of prognosis by combining N stage, SII and LDH was constructed to compare the prognosis of patients in high risk group, middle risk group and low risk group, and the receiver operating characteristic (ROC) curve analysis was used to evaluate its prognostic value.Results:The optimal cutoff value of SII is 447.2×10 9/L for predicting the 5-year overall survival (OS) of NPC patients, and the best cutoff value of LDH is 198.9 U/L. The proportion of patients with stage T3-4 and stage III-IVB in high SII group was higher than that in low SII group ( P<0.001). Multivariate Cox regression analysis showed that N stage, SII and LDH were independent factors of OS, progression-free survival (PFS) and distant metastasis-free survival (DMFS) of NPC patients (N stage, HR=1.705, 95% CI: 1.247-2.332; HR=1.755, 95% CI: 1.342-2.295; HR=2.161, 95% CI: 1.515-3.082. SII, HR=1.525, 95% CI: 1.097-2.119; HR=1.518, 95% CI: 1.150-2.004; HR=1.837, 95% CI: 1.272-2.653. LDH, HR=2.041, 95% CI: 1.403-2.968; HR=1.725, 95% CI: 1.233-2.414; HR=2.492, 95% CI: 1.690-3.672, respectively). After PSM, SII was still an independent prognostic factor of OS, PFS and DMFS in NPC patients ( HR=1.52, 95% CI: 1.09-2.12; HR=1.52, 95% CI: 1.15-2.00; HR=1.82, 95% CI: 1.26-2.63, respectively). Combined with N 2-3 stage, SII (>447.2×10 9/L), and LDH (>198.9 U/L), patients were divided into high-(3 risk factors), intermediate- (2 risk factors) and low-risk (0-1 risk factors) groups. The 5-year OS rates of patients in low-, intermediate- and high-risk groups were 86.1%, 79.8% and 41.2% respectively, the 5-year PFS rates were 80.7%, 70.2% and 33.9% respectively, and the 5-year DMFS rates were 88.9%, 79.2% and 47.5% respectively. There were significant differences in OS, PFS and DMFS among these three groups ( P<0.001). Distant metastasis was the main failure pattern in low-, intermediate- and high-risk groups, and the highest rate of distant metastasis was 83.3% (15/31) in high-risk group. ROC curve of the risk stratification model for predicting 5-year OS of NPC patients is 0.610, which is higher than TNM stage (0.609), SII (0.574) and LDH (0.558). Conclusions:Pretreatment SII and LDH are significantly correlated with the prognosis of patients with non-metastatic NPC. The combination of SII, LDH and N stage can stratify the prognostic risk of NPC patients. The risk stratification model can enhance the accuracy of prognosis.