Objective:To explore the feasibility and clinical value of sentinel lymph node (SLN) biopsy through cervix-uterine combined two-step injection with two tracers in patients with early stage endometrial cancer.Methods:From July 2019 to April 2021, a total of 73 patients, aged (54.2±3.3) year, who were preoperatively diagnosed as stage Ⅰ-Ⅱ endometrial cancer (including 56 low-risk patients and 17 medium-high risk patients) in Affiliated Hospital of Qingdao University were selected. According to the different sites of tracer injection, the patients were randomly divided into three groups: cervical injection group (25 cases): 1 ml of nano-carbon was used to inject at 3 and 9 o'clock in the cervix; uterine injection group (21 cases): the magnetic resonance imaging examination was performed to determine the location of the lesion, and 4 ml of methylene blue was injected into the uterine body at 2 sites where the lesion was located; combined injection group (27 cases): cervical injection of nano-carbon (1 ml) combined with uterine injection of methylene blue (4 ml). The SLN in all patients were identified under laparoscopy, removed, and followed by frozen pathological examination. Pathological ultra-staging was performed if the postoperative pathological outcome of SLN was negative. The total detection rate of SLN, bilateral pelvic SLN detection rate, sensitivity, negative predictive value, and location of SLN in each group were calculated and compared.Results:(1) In 73 patients with endometrial cancer, the overall detection rate of SLN was 88% (64/73), the detection rate of bilateral pelvic SLN was 67% (49/73), and the detection rate of para-aortic SLN was 49% (36/73). The overall detection rate of SLN (71%, 15/21) and bilateral pelvic SLN (43%, 9/21) in the intrauterine injection group were significantly lower than those in the cervical injection group [92% (23/25), 76% (19/25), respectively] and the combined injection group [96% (26/27), 78% (21/27), respectively; all P<0.05]; the detection rate of para-aortic SLN in the cervical injection group (28%, 7/25) was significantly lower than those in the intrauterine injection group and combined injection group [52% (11/21) and 67% (18/27), respectively; both P<0.05]. Among 73 cases with endometrial cancer, 9 had lymph node metastasis confirmed by postoperative pathological examination, 8 of them had lymph node metastasis detected by SLN and 1 had no lymph node metastasis detected by SLN, with a total sensitivity of 89% and a negative predictive value of 98%. The sensitivity and negative predictive value of cervical injection group and combined injection group were 100%, while the sensitivity and negative predictive value of intrauterine injection group were 67% and 95%. Among 56 low-risk patients, only one patient with lymph node metastasis was confirmed by postoperative pathology by SLN detection, and the metastasis rate was 2% (1/56), and the sensitivity and negative predictive value were 100%. Lymph node metastasis was confirmed in 8 of 17 patients (8/17) with a sensitivity of 88% and a negative predictive value of 90%. (2) A total of 459 SLN were detected in 73 endometrial cancer patients, with the highest proportion of external iliac (33.3%, 153/459).The obturator foramen was 25.3% (116/459), para-aortic 19.6% (90/459), iliac 12.0% (55/459), and presacral 9.8% (45/459). The proportion of para-aortic SLN in the cervical injection group was 12.4% (21/169), which were significantly lower than that in the intrauterine injection group and the combined injection group [27.4% (26/95) and 22.1% (43/195), respectively; both P<0.05]. (3) Pathological super-staging results: among 64 patients with negative SLN routine paraffin pathology, 4 cases of lymph node micro-metastases and 1 case of isolated tumor cell metastasis were detected, and the SLN micro-metastases rate was 8% (5/64), including 2 cases of low-risk patients and 3 cases of medium-high risk patients. Conclusions:SLN biopsy has high sensitivity and negative predictive value in patients with early endometrial cancer and could be used as an alternative to systematic lymph node dissection in low-risk patients. The SLN mapping through cervical-uterine combined injection could further improve the detection rate effectively and avoid the missed detection of positive para-aortic lymph node, especially for high-risk patients or patients with fundal tumor involvement.

Objective:To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC).Methods:(1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD 133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD 63 and CD 9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD 133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results:(1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD 133 positive ( CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% ( t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD 63 and CD 9 were positive. (3) Three groups′ OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) μm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD 133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD 133 was higher than that in the control group( F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant ( F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant ( F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups ( F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased ( P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group ( t=23.202, P<0.05). Conclusions:Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.

OBJECTIVE：To construct the evaluation system for TCM pharmaceutical care quality in public hospitals of Jilin province，and to provide reference for improving the quality of TCM pharmaceutical care in public hospitals. METHODS ：On Nov. 2020，20 relevant personnel and 10 experts in the field of TCM in Jilin province were selected by theoretical sampling method and objective sampling method ，respectively；grounded theory and Delphi method were adopted to sort out and score the evaluation system of TCM pharmaceutical care quality in public hospitals. Analytic hierarchy process （AHP）was used to determine the weight of each index. RESULTS ：In the two rounds of evaluation ，experts’enthusiasm，authority and opinion coordination were relatively high. Finally ，the evaluation system of TCM pharmaceutical care quality in public hospitals of Jilin Province was constructed ， consisting of 5 criterion layer indexes and 27 field layer indexes. The weights of the five criterion layer indexes from high to low are 0.290 for TCM pharmaceutical care management ，0.283 for TCM pharmaceutical care equipment ，0.163 for TCM prescription rationality，0.150 for TCM quality management ，0.144 for TCM pharmaceutical care ability. CONCLUSIONS ：The evaluation system of TCM pharmaceutical care quality in public hospitals of Jilin province established in this study is scientific and applicable ， and provides a basis for improving the quality of TCM pharmaceutical care in public hospitals. Public hospitals can set up priority improvement dimensions according to the results of the quality evaluation system ，and focus the limited human and material resources on priority improvement indexes.

Thimerosal has been widely used as a preservative in drug and vaccine products for decades.Due to the strong propensity to modify thiols in proteins,conformational changes could occur due to covalent bond formation between ethylmercury(a degradant of thimerosal)and thiols.Such a conformational change could lead to partial or even complete loss of desirable protein function.This study aims to investigate the effects of thimerosal on the capsid stability and antigenicity of recombinant human papillomavirus(HPV)18 virus-like particles(VLPs).Dramatic destabilization of the recombinant viral capsid upon thimerosal treatment was observed.Such a negative effect on the thermal stability of VLPs preserved with thimerosal was shown to be dependent on the thimerosal concentration.Two highly neutralizing antibodies,13H12 and 3C3,were found to be the most sensitive to thimerosal treatment.The kinetics of antigenicity loss,when monitored with 13H12 or 3C3 as probes,yielded two distinctly different sets of kinetic parameters,while the data from both monoclonal antibodies(mAbs)followed a biphasic expo-nential decay model.The potential effect of thimerosal on protein function,particularly for thiol-containing proteinaceous active components,needs to be comprehensively characterized during formulation development when a preservative is necessary.

Objective: To evaluate the effects of repeated ultraviolet A (UVA) radiation on DNA damage, repair and replication processes in human skin fibroblasts, and to explore their mechanisms. Methods: Fibroblasts were isolated from the circumcised foreskins of 3 children in the Department of Urological Surgery, Third Affiliated Hospital, Sun Yat-sen University, and subjected to a primary culture. Cultured human skin fibroblasts of 3rd-10th passages were divided into 2 groups: UVA group treated with repeated UVA radiation to establish a chronic photodamaged cell model, and control group receiving no treatment. Cell counting kit 8 (CCK-8) assay, β-galactosidase staining and flow cytometry were performed to assess cellular proliferative activity, and determine the proportion of photoaged cells and apoptosis rate respectively. Reverse transcription reaction was performed to establish a differentially expressed cDNA library, which was then subjected to high-throughput sequencing. The cDNA sequencing results were compared between the control group and UVA group, and the differentially expressed genes were analyzed in Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The data were compared between the two groups by using two independent sample t test. Results: Compared with the control group, the UVA group showed significantly decreased cellular proliferative activity (72.0% ± 5.2% vs. 96.0% ± 3.7%, t = 6.51, P < 0.05) , but significantly increased proportion of photoaged cells (79.7% ± 5.2% vs. 6.4% ± 0.8%, t=24.12, P < 0.05) and apoptosis rate (29.0% ± 3.3% vs. 6.0% ± 5.9%, t= 5.89, P < 0.05) . Among the key enzymes involved in DNA mismatch repair, replication and base excision repair processes, the expression of DNA ligase 1 (Lig1) , ribonuclease (RNase) H2A and helicase Dna2 in the UVA group was 0.47 ± 0.13, 0.44 ± 0.07 and 0.49 ± 0.11 times (all P < 0.01) that in the control group respectively. After the UVA-induced chronic photodamage in the human skin fibroblasts, After the UVA-induced chronic photodamage in the human skin fibroblasts, Lig expression decrease could block DNA single-base excision repair, Lig and Lig1 expression decrease could block DNA multiple-base excision repair, Lig1 expression decrease could block DNA mismatch repair, and RNaseH2A, Dna2 and Lig1 expression decrease could block DNA replication. Conclusion Repeated UVA radiation can change the expression of key enzymes involved in DNA base excision repair, DNA mismatch repair and DNA replication processes in skin fibroblasts, and then affect DNA repair and DNA replication processes in skin fibroblasts.

Objective:To summarize the evidence for the link between rheumatoid arthritis and risk of vertebral fractures or vertebral deformities with a meta-analysis, so as to provide objective proof for early preventing and the development of vertebral deformity and fractures.Methods:Wanfang,CNKI,VIP,PUBMED,Springlink and Elsevier were retrieved for all publications relating to rheumatoid arthritis and vertebral fractures in women.According to inclusion and exclusion criteria,two investigators collected their data individually,then statistical analysis was performed using Stata 12.0 software.Results: Eight case-control studies were enrolled, including 86 741 participants,2 258 of them with RA.The results of Meta-analysis showed that a higher incidence of vertebral fractures in RA,and Odds ratio was 3.70 with a 95%confidence interval(2.47-5.55,P<0.000 1).The publication bias analysis did not reveal any evidence of obvious asymmetry, and the sensitivity analysis showed that omission of any individual study made no significant difference for all comparison models,suggesting that our results were statistically robust.Conclusion:RA may be one of the risk factors for the vertebral fractures.

Objective To study the ultrasonic extraction technology of tyrosol in Rhodiolae Crenulatae Radix et Rhizoma;To mathematically simulate the extraction process.Methods The content of tyrosol was set as index;HPLC was used;the ultrasonic extraction process of tyrosol in Rhodiolae Crenulatae Radix et Rhizoma was optimized by an orhogonal experiment and mathematic simulation.Results The optimum ultrasonic extraction conditions were as follows:80%ethanol was used as extraction solvent, the ratio of material to liquid was 1:35 (g:mL), with the extraction time of 30 min and ultrasonic power of 240 W. In these conditions, the extracting rate of tyrosol was 0.150 1%. Compared with the heating reflux method, the extraction time should be shortened by 66.7% and the extracting rate should be increased by 12%.Conclusion The extraction method is simple and the extraction rate of effective components is high. Mathematical simulation values based on ultrasonic extraction are consistent with experiment values.

OBJECTIVE:To optimize the ultrasonic extraction technology of salidroside in Rhodiola rosea. METHODS:With the content of salidroside as the index,L16(45)orthogonal test was designed to observe the effects of 4 factors including the mass fraction of ethanol,solid-liquid ratio,extraction time and ultrasonic power on the content of salidroside in R. rosea extraction solu-tion. By using Matlab 6.5 software,mathematical simulation of ultrasonic extraction process of salidroside was made with the data of orthogonal test. Multiple regression analysis method was adopted to optimize the ultrasonic extraction technology of salidroside, and then the extraction result of optimal technology and that of traditional heating reflux technology were compared. RESULTS:The optimal extraction technology of salidroside was as follows as ethanol mass fraction of 77%,solid-liquid ratio of 1∶34 (g∶ml),extraction time of 34 min,ultrasonic power of 237 W(ultrasonic frequency of 40 kHz). Under the above-mentioned condi-tions,the content of salidroside was up to 0.982 4%,close to the predicted value of 0.989 4%. Compared to heating reflux meth-od,the ultrasonic method has similar content of salidroside but extraction time was shortened by 62.2%. CONCLUSIONS:The ul-trasonic extraction method for extracting the salidroside in R. rosea is simple,requires shorter time,and giving rise to higher extrac-tion rate.

BACKGROUND:The development of keloid is a progress of fibrosis in wound healing, and involves various fibrosis-related cytokines. Bone morphogenetic protein 7 (BMP7), Gremlin and high mobility group box-1 (HMGB1) play an important role in fibrosis of many organs, but their role in keloid tissue has rarely been reported.
OBJECTIVE:To investigate the role of BMP7, Gremlin, vascular endothelial growth factor (VEGF) and HMGB1 in the development of keloid.
METHODS:The protein levels and distribution of BMP7, Gremlin, VEGF and HMGB1 in 20 cases of keloid and 20 cases of normal skin were detected by immunohistochemistry and western blot analysis, respectively. And the correlations among expression levels of BMP7, Gremlin, VEGF and HMGB1 in keloid were analyzed.
RESULTS AND CONCLUSION:In keloid tissue, the expression levels of Gremlin, VEGF and HMGB1 were significantly higher than that in normal skin (P<0.01), while the expression levels of BMP7 were significantly lower (P<0.01). The levels of Gremlin were negatively correlated with the levels of BMP7 (r=-0.539, P<0.05). And the levels of VEGF were positively correlated with the levels of HMGB1 (r=0.56, P<0.05). The overexpression of Gremlin and decreased expression of BMP7, as wel as the increased expression of HMGB1 and VEGF, may contribute to the pathogenesis of fibrosis in the development of keloid.

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-- Patchedl (PTCH1) and Glil in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Glil in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEPI), re-spectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concen-trations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expres-sion of PTCH1 and Gill was detected in both NB samples and S-type NB cell lines. Cyclopamine de-creased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopaminc could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase, while Shh induced the reverse effects and could partially pre-vent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies re-vealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic ap-proach to NB.