1.Investigation of the immune profile of multiple myeloma patients achieving long-term survival after autologous stem cell transplantation
Jingli GU ; Chuhang ZHONG ; Meilan CHEN ; Lifen KUANG ; Xiaozhe LI ; Beihui HUANG ; Junru LIU ; Juan LI
Chinese Journal of Internal Medicine 2024;63(4):365-370
Objective:To identify the characteristics of the bone marrow immune microenvironment associated with long-term survival in multiple myeloma (MM) patients.Methods:In the follow-up cohort of patients with newly diagnosed MM and who received “novel agent induction therapy and subsequent autologous stem cell transplantation and immunomodulator maintenance therapy” in the First Affiliated Hospital of Sun Yat-sen University, a cross-sectional study was carried out between August 2019 and May 2020. Using NanoString technology, the RNA expression of 770 bone marrow immune-related markers was compared between 16 patients who had progression-free survival ≥5 years and 5 patients with progressive disease. Among the 16 patients who achieved long-term survival, 9 achieved persistent minimal residual disease (MRD) negative while the other 7 had persistent positive MRD. The functional scores of each kind of immune cells were calculated based on the expression level of characteristic genes, so as to indirectly obtained the proportion of each immune cell subset. The Mann-Whitney U test and the Kruskal Wallis test were used for statistical analysis. Results:The proportion of neutrophils was significantly higher in long-surviving MM patients than in patients with progressive disease [functional scores, 13.61 (13.33, 14.25) vs. 12.93 (12.58, 13.38); Z=2.31, P=0.021]. Among long-surviving patients, those who were MRD-positive had a significantly greater number of mast cells compared with those who were MRD-negative [functional scores, 7.09 (6.49, 8.57) vs. 6.03 (5.18, 6.69); H=2.18, P=0.029]. Compared with patients with progressive disease, four genes (CTSG, IFIT2, S100B, and CHIT1) were significantly downregulated and six (C4B, TNFRSF17, CD70, IRF4, C2, and GAGE1) were upregulated in long-surviving patients. Among long-surviving patients, only gene CMA1 was significantly upgraded, 10 genes (ISG15, OAS3, MX1, IFIT2, DDX58, SIGLEC1, CXCL10, IL1RN, SERPING and TNFSF10) were significantly downregulated in the MRD-positive group compared with that in the MRD-negative group, the first 5 of which are related to the interferon response pathway. Conclusions:The increased neutrophil and mast cell numbers may be related to long-term survival in MM. Interferon signaling activation may be a key bone marrow immune profiling feature for MRD-negative, long-surviving patients with MM.
2.Epidemiological features of pathogens of acute respiratory tract infection in children in Guangzhou area from 2018 to 2021
Jiahui XIE ; Lu KUANG ; Jiayu ZHONG ; Yingying ZHANG ; Yi CHEN ; Xiaomin LIU ; Wanli LIANG ; Fuying CHEN ; Minzhang TAN ; Bing ZHU
Chinese Journal of Infectious Diseases 2023;41(2):137-143
Objective:To investigate the epidemiology of pathogens of acute respiratory tract infection (ARTI) in children in Guangzhou area.Methods:A total of 13 610 hospitalized children with ARTI in Guangzhou Women and Children′s Medical Center from January 2018 to December 2021 were enrolled. Throat swab specimens were collected, and fluorescent quantitative polymerase chain reaction (PCR) was performed to detect 11 respiratory pathogens, including respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV), human rhinovirus (HRV), human bocavirus (HBoV), human metapneumovirus (HMPV), enterovirus (EV), influenza A virus (IFA), influenza B virus (IFB), Mycoplasma pneumoniae (MP) and Chlamydia pneumoniae (CP). Grouping according to age (< one year group, one to < three years group, three to < six years group, six to 14 years group) and season. Chi-square test was used for statistical analysis. Results:At least one pathogen was detected in 6 331 cases among 13 610 patients, and the overall positive rate was 46.52%. The detection rates from high to low were as follows: RSV (13.75%(1 872/13 610)), ADV (4.82%(656/13 610)), PIV (4.82%(656/13 610)), MP (4.54%(618/13 610)), HRV (3.39%(462/13 610)), HBoV (2.64%(359/13 610)), HMPV (2.59%(352/13 610)), EV (1.76%(239/13 610)), IFA (1.29%(176/13 610)), IFB (0.90%(122/13 610)) and CP (0.30%(41/13 610)). The positive rate of viral detection showed significant differences among different age groups ( χ2=49.91, P<0.001), and the highest positive rate was in the age group of one to
3.Preparation of silk fibroin/hyaluronic acid composite hydrogel based on thiol-ene click chemistry.
Leidan CHEN ; Mingqiang ZHONG ; Jinyi CHEN ; Zhenjie LIU ; Tairong KUANG ; Tong LIU ; Feng CHEN
Journal of Zhejiang University. Medical sciences 2023;52(3):285-295
OBJECTIVES:
To design and prepare silk fibroin/hyaluronic acid composite hydrogel.
METHODS:
The thiol modified silk fibroin and the double-bond modified hyaluronic acid were rapidly cured into gels through thiol-ene click polymerization under ultraviolet light condition. The grafting rate of modified silk fibroin and hyaluronic acid was characterized by 1H NMR spectroscopy; the gel point and the internal microstructure of hydrogels were characterized by rheological test and scanning electron microscopy; the mechanical properties were characterized by compression test; the swelling rate and degradation rate were determined by mass method. The hydrogel was co-cultured with the cells, the cytotoxicity was measured by the lactate dehydrogenase method, the cell adhesion was measured by the float count method, and the cell growth and differentiation on the surface of the gel were observed by scanning electron microscope and fluorescence microscope.
RESULTS:
The functional group substitution degrees of modified silk fibroin and hyaluronic acid were 17.99% and 48.03%, respectively. The prepared silk fibroin/hyaluronic acid composite hydrogel had a gel point of 40-60 s and had a porous structure inside the gel. The compressive strength was as high as 450 kPa and it would not break after ten cycles. The water absorption capacity of the composite hydrogel was 4-10 times of its own weight. Degradation experiments showed that the hydrogel was biodegradable, and the degradation rate reached 28%-42% after 35 d. The cell biology experiments showed that the cytotoxicity of the composite gel was low, the cell adhesion was good, and the growth and differentiation of the cells on the surface of the gel were good.
CONCLUSIONS
The photocurable silk fibroin/hyaluronic acid composite hydrogel can form a gel quickly, and has excellent mechanical properties, adjustable swelling rate and degradation degree, good biocompatibility, so it has promising application prospects in biomedicine.
Fibroins/chemistry*
;
Hydrogels/chemistry*
;
Hyaluronic Acid/chemistry*
;
Biocompatible Materials/chemistry*
;
Click Chemistry
;
Sulfhydryl Compounds
;
Silk/chemistry*
4. Apigenin inhibits liver fibrosis through PDK1/AKT signaling pathway in mice
Xin-Dong CHEN ; Pei-Yao YAN ; Jia-Ni KUANG ; Tao SUN ; Biao HUANG ; Yuan QIN ; Wei-Long ZHONG ; Kai-Liang QIAO
Chinese Pharmacological Bulletin 2022;38(7):1010-1016
Aim To evaluate the therapeutic effect of apigenin on liver fibrosis in mice anrl the pharmacologi¬cal mechanism.Methods Carbon tetrachloride ( CC14) -induced liver fibrosis mouse model was estab¬lished.The mice were divided into six groups of con¬trol, model, silibinin(55 mg • kg 1 • d 1 ) , apigenin in high dosage (60 mg • kg 1 • d 1 ) , apigenin in mid¬dle dosage( 30 mg • kg 1 • d 1 ) and apigenin in low dosage( 15 mg • kg 1 • d 1 ).The general life status, body weight and liver coefficient of the mice in every group were recorded.HE staining, Masson staining, immunohistochemistry and Western blot were used to e- valuate the effect of apigenin on the pathological chan¬ges, the markers related to epithelial-mesenchymal transition and signaling pathways of liver tissues.Re¬sults In CCI4-induced liver fibrosis mice, middle and high-dosage of apigenin could improve the general life status, increase body weight, decrease liver coeffi¬ cient, and significantly improve liver lesions.Middle and high-dosage of apigenin significantly increased the expression of the epithelial marker protein E-cadherin and significantly decreased the expression of the mes¬enchymal marker protein Vimentin in liver tissues of mice with the disease.The further results showed that middle and high-dosage apigenin could significantly in¬hibit the expression of phosphorvlated PDK1 and phos- phorvlated AKT protein in liver tissues of model mice.Conclusions Apigenin can inhibit EMT by inhibiting PDK1/AKT signaling pathway, which plays an anti-fi- brosis role.The apigenin has the potential to be further developed as a drug to protect the liver and treat liver fibrosis.
5.Ferulic Acid Promotes Longevity and Stress Tolerance via the Insulin/IGF Signaling Pathway and DAF-16 in Caenorhabditis Elegans
Qi-fei KUANG ; Qiao-chao CHEN ; Ling ZHANG ; Wen HE ; Zhong PEI
Journal of Sun Yat-sen University(Medical Sciences) 2021;42(2):193-201
ObjectiveTo investigate the effect on life extension and the mechanism of ferulic acid in Caenorhabditis elegans. MethodsWe used C. elegans as an anti-aging model, and different concentrations of ferulic acid (0, 1, 10, 50 mmol/L) were given to explore its regulatory effects on the lifespan, anti-stress ability, and lipofuscin level ofC. elegans. And we used qPCR to screen senescence related genes, and DAF-2 (DR1572) and DAF-16 (CF1038) mutants were used to investigate the potential mechanism of ferulic acid. ResultsCompared with the blank control group, ferulic acid significantly extended the lifespan of C. elegans and increased its antioxidant and thermal stress (P<0.001), inhibited the accumulation of lipofuscin related to aging (P < 0.001), and did not affect its fertility and body shape (P>0.05) . After feeding nematode in the 50 mmol/L ferulic acid group, mRNA expression levels of DAF-2, Akt-2 and AGE-1 genes were down-regulated (P<0.05), mRNA expression levels of DAF-16 genes were up-regulated (P<0.05) and the expression levels of its downstream target genes sod-3, ctl-1, clk-2 and dod-17 were activated (P<0.05). After 50 mmol/L ferulic acid treatment, the proportion of daf-16 ::GFP in the nematode cell nucleus increased from (5.3±1.5) % to (28±3) %. The lifespan of DAF-2 (DR1572) and DAF-16 (CF1038) mutant nematodes was not significantly extended after treatment with 50 mmol/L ferulic acid (P>0.05). ConclusionsFerulic acid can promote the nuclear localization of daf-16 through insulin /IGF signaling pathway, improve the stress resisting ability of the organism, and prolong the lifespan of nematode.
6.Anti?lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro
Jianfeng TAN ; Chenghua ZHONG ; Jun CHEN ; Quanwei GUO ; Jun KUANG ; Jianhua ZHANG
Chinese Journal of Oncology 2019;41(7):501-507
To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat?treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods Bone marrow cells were induced by the recombinant mouse fms?like tyrosine kinase receptor 3 ligand ( rmFlt3?L) in vitro, myeloid dendritic cells ( mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC ∶ pDC=1 ∶ 1 were stimulated with heat?treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ( MHC?Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin?12 (IL?12), interlukin?6 (IL?6), and tumor necrosis factor α ( TNF?α) were detected by enzyme?linked immunosorbent assay ( ELISA). Results The lymphocyte proliferation in mice stimulated with mDC+pDC group loaded with heat?treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively ( P<0.05). When the ratio of effector cells:target cells (E ∶ T) was 10 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E ∶ T was 20 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 54.77%± 3.28%, significantly higher than 35.25%± 1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E ∶ T was 40 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 73.01%± 0.91%, significantly higher than 51.36%± 0.58% of mDC group and 22.65%± 1.28% of pDC group, respectively (P<0.05 ). With the rate of E ∶ T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC?Ⅱ of mDC:pDC=1 group pulsed with heat?treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL?6 cytokine concentrations of mDC+pDC group, mDC group and pDC group loaded with heat?treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67± 16.07) pg/ml, respectively. The concentrations of IL?12 in each group were ( 2 568.75± 119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF?α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL?6, IL?12 and TNF?α secreted by mDC+pDC group were much higher than those of mDC group and pDC group under the same culture conditions ( P<0.05). Conclusions The mDCs and pDCs combined vaccines pulsed with heat?treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti?tumor effect is related with up?regulation of co?stimulatory molecules and increased secretion of cytokines.
7. Anti-lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro
Jianfeng TAN ; Chenghua ZHONG ; Jun CHEN ; Quanwei GUO ; Jun KUANG ; Jianhua ZHANG
Chinese Journal of Oncology 2019;41(7):501-507
Objective:
To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice.
Methods:
Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA).
Results:
The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (
8. Analysis of causes and treatment methods of complication of early acute kidney injury in four severely burned patients
Bin CHEN ; Fang KUANG ; Xiaojian LI ; Zhi ZHANG ; Zhongyuan DENG ; Xuhui ZHANG ; Tao ZHANG ; Xiaomin ZHONG ; Wenbin TANG ; Changling LIU
Chinese Journal of Burns 2019;35(2):110-115
Objective:
To analyze the causes of complication of early acute kidney injury (AKI) in four severely burned patients, and to explore the related treatment methods.
Methods:
The clinical data of 4 patients with severe burn complicated with early AKI admitted to Guangzhou Red Cross Hospital Affiliated to Medical College of Jinan University (hereinafter referred to as our hospital) from June 2014 to December 2017 were retrospectively analyzed. All the patients were male, aged 23-33 (30±5) years old, with depth of burns ranged from deep partial-thickness to full-thickness, complicated with myofascial compartment syndrome of extremities and varying degrees of striated muscle injury, and treated in other hospitals before transfer to our hospital. The patients were numbered from small to large according to the total burn area. The total burn area of patients No. 1, 2, 3, and 4 was 10%, 80%, 90%, and 95% total body surface area respectively, their occurrence time of early AKI was 48, 11, 29, and 48 hours after injury respectively, and their time of arriving our hospital was 60, 11, 29, and 144 hours after injury respectively. Hypovolemic shock occurred in patients No. 2 and 3 at admission to our hospital. All the patients received continuous renal replacement therapy (CRRT) after admission to our hospital. Under the support of hemodynamic monitoring and organ function monitoring, the limbs complicated with myofascial compartment syndrome were incised, thorough decompression exploration was performed, and necrotic muscle tissue was removed or amputation was performed. After escharectomy and decompression of limbs, fresh granulation wounds were formed by temporarily covering wounds with Jieya dressing skin or pig skin, multiple debridements, and vacuum sealing drainage. Fresh granulation wounds and other wounds underwent staged eschar excision and shaving were covered with autologous Meek skin graft, particulate skin graft, reticular skin graft and small skin graft respectively. The treatment outcome, CRRT time, operation times, time of recovery of serum creatinine and myoglobin, length of hospital stay, and follow-up were recorded.
Results:
All the 4 patients were cured after transfer to our hospital. Among them, totally 5 limbs of patients No. 1 and No. 4 underwent amputation because of complication of myofascial compartment syndrome and a large amount of necrotic muscle which could not be preserved. Patients No. 1, 2, 3, and 4 were treated with CRRT for 19, 35, 14, and 25 days respectively and performed with operation for 5, 6, 10, 8 times respectively. Serum creatinine of patients No. 1, 2, 3, and 4 returned to normal on 22, 35, 37, and 48 days after transfer respectively, and their serum myoglobin returned to normal on 18, 28, 25, and 30 days after transfer respectively. Patients No. 1, 2, 3, and 4 were hospitalized for 52, 105, 148, and 156 days and discharged after basic wound healing. Follow-up for 1 to 36 months showed no abnormal renal function in 4 patients.
Conclusions
The early AKI in patients No. 1 and 4 was caused by rhabdomyolysis after severe burn complicated with myofascial compartment syndrome, while that of the other 2 cases were also related to hypovolemic shock and poor renal perfusion. The success rate of early AKI treatment in severely burned patients can be effectively improved by removing the causes of diseases at the same time of CRRT and actively treating burn wounds under the support of organ function and hemodynamic monitoring.
9.Anti?lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro
Jianfeng TAN ; Chenghua ZHONG ; Jun CHEN ; Quanwei GUO ; Jun KUANG ; Jianhua ZHANG
Chinese Journal of Oncology 2019;41(7):501-507
To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat?treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods Bone marrow cells were induced by the recombinant mouse fms?like tyrosine kinase receptor 3 ligand ( rmFlt3?L) in vitro, myeloid dendritic cells ( mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC ∶ pDC=1 ∶ 1 were stimulated with heat?treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ( MHC?Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin?12 (IL?12), interlukin?6 (IL?6), and tumor necrosis factor α ( TNF?α) were detected by enzyme?linked immunosorbent assay ( ELISA). Results The lymphocyte proliferation in mice stimulated with mDC+pDC group loaded with heat?treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively ( P<0.05). When the ratio of effector cells:target cells (E ∶ T) was 10 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E ∶ T was 20 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 54.77%± 3.28%, significantly higher than 35.25%± 1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E ∶ T was 40 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 73.01%± 0.91%, significantly higher than 51.36%± 0.58% of mDC group and 22.65%± 1.28% of pDC group, respectively (P<0.05 ). With the rate of E ∶ T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC?Ⅱ of mDC:pDC=1 group pulsed with heat?treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL?6 cytokine concentrations of mDC+pDC group, mDC group and pDC group loaded with heat?treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67± 16.07) pg/ml, respectively. The concentrations of IL?12 in each group were ( 2 568.75± 119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF?α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL?6, IL?12 and TNF?α secreted by mDC+pDC group were much higher than those of mDC group and pDC group under the same culture conditions ( P<0.05). Conclusions The mDCs and pDCs combined vaccines pulsed with heat?treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti?tumor effect is related with up?regulation of co?stimulatory molecules and increased secretion of cytokines.
10. Pathological insights of radiotherapy-related damage to surgical margin after preoperative radiotherapy in patients with rectal cancer
Qinghua ZHONG ; Peihuang WU ; Qiyuan QIN ; Yingyi KUANG ; Tenghui MA ; Huaiming WANG ; Yaxi ZHU ; Daici CHEN ; Jianping WANG ; Lei WANG
Chinese Journal of Surgery 2017;55(7):507-514
Objective:
To investigate the effect of irradiation to anastomosis from preoperative radiotherapy for patients with rectal cancer by studying the pathological changes.
Methods:
In this retrospective study, patients enrolled in the FOWARC study from January 2011 to July 2014 in the Sixth Affiliated Hospital of Sun Yat-Sen University were included. In the FOWARC study, enrolled patients with local advanced rectal cancer were randomly assigned to receive either neoadjuvant chemo-radiotherapy or chemotherapy. Among these patients, 23 patients were selected as radiation proctitis (RP)group, who fulfilled these conditions: (1) received neoadjuvant chemo-radiotherapy followed by sphincter-preserving surgery; (2) developed radiation proctitis as confirmed by preoperative imaging diagnosis; (3) had intact clinical samples of surgical margins. Twenty-three patients who had received neoadjuvant chemo-radiotherapy but without development of radiation proctitis were selected as non-radiation proctitis (nRP) group. Meanwhile, 23 patients received neoadjuvant chemotherapy only were selected as neoadjuvant chemotherapy (CT) group. Both nRP and CT cases were selected by ensuring the basic characteristics such as sex, age, tumor site, lengths of proximal margin and distal margin all maximally matched to the RP group. Both proximal and distal margins were collected for further analysis for all selected cases. Microscopy slices were prepared for hematoxylin & eosin staining and Masson staining to show general pathological changes, and also for immunohistochemistry with anti-CD-34 as primary antibody to reveal the microvessel. Microvessel counting in submucosal layer and proportion of macrovessel with stenosis were used to evaluate the blood supply of the proximal and distal end of anastomosis. A modified semi-quantitative grading approach was used to evaluate the severity of radiation-induced injury. Either ANOVA analysis, Kruskal-Wallis rank-sum test or χ2 test was used for comparison among three groups, and Mann-Whitney

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