Objective: To provide a method of microbial limit test for Changyanning tablets and carry out the verification of the mothod.Methods: A plate method was used in the total aerobic microbial count and the concentration of the test liquid was 1∶100.A plate method was used in the total yeast and mold count while the concentration of the test liquid was 1∶10.The direct inoculation method was used for the detection of Escherichia coli,bile-tolerant gram-negative bacteria and Salmonella enterica subsp.Results: The recoveries of the five strains were from 0.5 to 2.0.Conclusion: The method is suitable for the microbial limit examination of Changyanning tablets.

Objective To explore the clinical effect and safety of carrellizumab combined with albumin-bound paclitaxel on the treatment of patients with locally advanced esophageal cancer.Methods Ninety-eight patients with locally advanced esophageal cancer were randomly divided into the study group and the reference group,with 49 cases in each group.The reference group was treated with albumin-bound paclitaxel,while the study group was treated with camrelizumab+albumin-bound paclitaxel.After treatment,the overall efficacy was evaluated,and the disease control rate was calculated.Serum tumor markers[high mobility group protein B1(HMGB1),squamous cell carcinoma associated antigen(SCCA)],PD-1 and PD-L1 levels were detected by enzyme-linked immunosorbent assay(ELISA)before and after treatment.Immunohistochemistry was used to detect the microvascular density(MVD)and the expression levels of PD-1 and PD-L1 in esophageal carcinoma tissue.The percentages of CD3+,CD4+and CD8+cells were detected by flow cytometry.Patients were followed up and adverse reactions and survival were recorded.Results The disease control rate was higher in the study group than that of the control group(P＜0.05).There were no significant differences in all indexes between the groups before treatment(P＞0.05).After treatment,HMGB1 level,SCCA level,MVD,serum PD-1 level and tumor tissue high expression rate of PD-1 were all decreased in the two groups,while serum PD-L1 level,PD-L1 high expression rate and percentages of CD3+,CD4+and CD8+cells were all increased(P＜0.05).Except there was no significant difference in MVD between groups,the other indexes were significantly changed in the study group compared with the control group(P＜0.05).There was no significant difference in the incidence of adverse reactions between the two groups(P＞0.05).The progression-free survival rate was higher in the study group than that of the control group(P＜0.05).Conclusion Carrellizumab combined with albumin-bound paclitaxel in the treatment of locally advanced esophageal cancer can help control lesion progression and inhibit angiogenesis,with good safety.

Objective:To compare the efficacy and safety of robot-assisted laparoscopic and laparoscopic partial nephrectomy (RAPN and LPN) for patients with completely endophytic renal tumor.Methods:A total of 73 patients with completely endophytic renal tumor receiving RAPN (n=29) or LPN (n=44) in our center between January 2015 and June 2021 were retrospectively collected. There were 21 males and 8 females in RAPN group. The average age was 48.6±13.7 years old, average tumor size was 2.9±0.9 cm with 13 left tumors and 16 right tumors, average R. E.N.A.L. score was 9.2±1.0, and average preoperative eGFR was 82.6±10.7 ml/(min·1.73 m 2). There were 27 males and 17 females in LPN group. The average age was 50.1±12.3 years old, average tumor size was 2.9±0.9 cm with 24 left tumors and 20 right tumors, average R. E.N.A.L. score was 9.1±1.3, and average preoperative eGFR was 81.7±9.6 ml/(min·1.73 m 2). There was no significant difference in above variables between two groups. The operative time, warm ischemia time, blood loss, postoperative complication, postoperative hospital stay and postoperative 3 months renal function of two groups were compared. Results:All 73 patients successfully underwent RAPN or LPN and no patient converted to radical nephrectomy or open surgery. There was no significant difference in operation time [140(80, 160) min vs. 150 (90, 180) min, P=0.264], intraoperative estimated blood loss[150 (100, 200)vs. 180 (120, 200) ml, P=0.576]and postoperative hospital stay (7.0±2.7 vs. 7.4±2.1 days, P=0.480) between two groups. Compared with LPN group, RAPN group had obvious less warm ischemia time (23.1±3.3 vs. 27.6±4.7 min, P<0.001). No obvious complication occurred in RAPN group and one case with postoperative hemorrhage occurred in LPN group. No positive margin occurred in either group. There was no difference in renal function 3 months after operation between the two groups [73.2±6.3 vs.70.5±7.6ml/(min·1.73 m 2), P=0.117]. The median follow-up period was 22.6 months with no tumor recurrence or metastasis. Conclusions:For experienced surgeons, both RAPN and LPN are safe and feasible for patients with completely endophytic renal tumor. Compared with LPN, RAPN has advantages of perioperative curative effect, which could reduce the operating difficulty and shorten the warm ischemia time.

Objective:To investigate the effect of astragaloside IV (AS-IV) regulating the signal axis of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) on the mobilization of bone marrow endothelial progenitor cells (EPCs) to peripheral blood in diabetes skin ulcer (DSU) rats.Methods:Twenty four SPF grade male Sprague Dawley (SD) rats were selected to make the model of type 2 diabetes rats by intraperitoneal injection of 30 mg/kg 1% (plastid ratio) streptozotocin, and then round full-thickness skin with a diameter of 2 cm was cut on both sides of the waist and back to make the skin ulcer model of diabetes rats. After that, they were randomly divided into AS-IV group (50 mg/kg AS-IV), blocker group (50 mg/kg AS-IV+ 5 mg/kg AMD3100) and model group. At the same time, a blank group ( n=8) was set up, The drug was administered via intraperitoneal injection, and the model group and blank group were treated with 0.9% NaCl of equal volume. On the 10th day, peripheral blood, femoral bone marrow, and wound neovascularization tissues of rats were collected. The number of EPCs in peripheral blood of each group of rats was measured by flow cytometry, and the protein expression of SDF-1α and CXCR4 in peripheral blood, femoral bone marrow, and wound neovascularization tissues of rats was detected by enzyme-linked immunosorbent assay (ELISA); At the same time, the wound healing rates of each group were tested. Results:On the 10th and 21st day after modeling, the wound healing rate of each group of rats was compared. The blank group healed the fastest, while the model group healed the slowest. The AS-IV group had better healing than the model group and the blocker group, and the difference was statistically significant (all P<0.05). On the 10th day after modeling, the positive rates of peripheral blood EPCs in the white group, AS-IV group, and blocker group were significantly higher than those in the model group (all P<0.05), while the positive rates of peripheral blood EPCs in the blocker group were significantly lower than those in the AS-IV group (all P<0.05). On the 10th day after modeling, the protein expression of SDF-1α and CXCR4 in the wound, serum, and bone marrow of the model group was the lowest, while the protein expression in the blank group was the highest (all P<0.05). The protein expression of SDF-1α and CXCR4 in the wound, serum, bone marrow of the AS-IV group was significantly higher than that of the blocker group and model group, and the difference was statistically significant (all P<0.05). Conclusions:Astragaloside IV can promote the mobilization and migration of endothelial progenitor cells from bone marrow to peripheral blood in diabetes ulcer rats by regulating SDF-1α/CXCR4 signal axis, and can participate in angiogenesis of diabetes ulcer wounds as seed cells to promote the healing of diabetes skin ulcers.

Objective:To investigate the effects of mesenchymal stem cells-derived exosomes (MSC-Exos) secreted by mesenchymal stem cells (MSCs) induced by astragaloside IV (AS-IV) on the biological function and pyroptosis of human umbilical vein endothelial cells (HUVECs) injured by high glucose.Methods:After human umbilical cord blood mesenchymal stem cells (hUCBMSCs) were intervened with 400 mg/L of AS-IV, exosomes were extracted, and then the morphology and specific markers of exosomes were identified. Human umbilical vein endothelial cells (HUVECs) were cultured in a medium with a glucose concentration of 30 mmol/L to prepare a high glucose-impaired HUVECs model. High glucose-impaired HUVECs were randomly divided into experimental and model groups, with the experimental group intervened with 100 μg/ml of MSC-Exos and the model group intervened with an equal volume of PBS solution, while a blank control group was also set up. Cell counting Kit-8 (CCK-8) cell proliferation assay, adhesion assay, matrigel tube formation assay and scratch assay were used to detect the effects of AS-IV-mediated MSC-Exos on the proliferation, adhesion, tube formation and migrationability of HUVECs; Western blot and real time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the protein and mRNA expression of scorch death-related molecules, such as Caspase-1, GSDMD (Gasdermin D) and NLRP3 in each group.Results:The proliferation, adhesion number, tube number and migration width of HUVECs cells were significantly lower than those in the blank group ( P<0.05); The expression of Caspase-1, GSDMD, NLRP3 protein and their mRNA increased significantly ( P<0.001); Under the intervention of MSC-Exos mediated by AS-IV, the cell proliferation, adhesion number, tube number and migration width of HUVECs were significantly higher than those in the model group ( P<0.05); The expression of Caspase-1, GSDMD, NLRP3 protein and their mRNA decreased, with statistically significant difference ( P<0.05). Conclusions:AS-IV mediated MSC-Exos can significantly improve the biological function of high glucose-impaired endothelial cells, and its mechanism may be related to anti-pyroptosis.

Objective To explore the effect of miR-296-3p on hepatic fibrosis induced by bile duct ligation(BDL)in rats.Methods 25 SD rats were randomly divided into sham group,model(BDL)group,NC adv group,miR-296-3p adv group and miR-296-3p sponge adv group,with 5 rats in each group.The pathological changes were ob-served in rat liver tissue via HE,Masson and Sirius Red staining;the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(TBIL)in the serum of rat in each group were detected;the expression levels of miR-296-3p,interleukin(IL)-6,IL-1 β,tumor necrosis factor(TNF)-α and smooth muscle actin(α-SMA),type Ⅰ collagen(Col1A1),and connective tissue growth factor(CTGF)mRNA in rat liver tissue were detected by qRT-PCR;the expression levels of α-SMA,Col1A1 and CTGF proteins were detected by Western blot.Immunohistochemical staining(IHC)was performed to detect the expression of α-SMA in liver tissue.Target genes of miR-296-3p was predicted by bioinformatic analysis using the online database.Zinc finger and BTB do-main-containing protein20(ZBTB20)mRNA and protein expression levels were detected.Results The pathologi-cal staining results showed that compared with sham group,a large number of infiltrated inflammatory cells and col-lagen deposition were observed in the liver tissues of rats in the BDL group and NC adv group.Compared with NC adv group,the inflammatory cells and collagen deposition decreased in the liver tissues of miR-296-3p adv group.However,in miR-296-3p sponge adv group,collagen product and inflammatory reaction increased.Compared with sham group,the contents of ALT,AST and TBIL in serum of rats in BDL group and NC adv group increased,the expression level of miR-296-3p decreased,the mRNA expression levels of IL-6,IL-1β,TNF-α increased,and the mRNA and protein expression levels of α-SMA,Col1A1 and CTGF increased(all P＜0.05).Compared with the NC adv group,the contents of ALT,AST and TBIL in serum of rats in miR-296-3p adv group decreased,the ex-pression level of miR-296-3p increased,the mRNA expression levels of IL-6,IL-1 β and TNF-α,and the mRNA and protein expression levels of α-SMA,Col1A1 and CTGF in liver tissues decreased(all P＜0.05).The results of miR-296-3p sponge adv group were opposite to those of miR-296-3p adv group(all P＜0.05).The bioinformat-ics website predicted that ZBTB20 might be a candidate target gene of miR-296-3p.Compared with sham group,the expression of ZBTB20 mRNA and protein in the liver tissues of BDL group and NC adv group increased(P＜0.05),and the expression of ZBTB20 in the liver tissues of miR-296-3p adv group decreased compared with NC adv group(P＜0.05).However,the expression of ZBTB20 in liver tissues of miR-296-3p sponge adv group in-creased(P＜0.05).Conclusion miR-296-3p expression decreases in BDL-induced hepatic fibrosis in rats,and miR-296-3p may inhibit hepatic fibrosis in BDL rats by targeting ZBTB20.

Objective To investigate the effect of miR-199a-5p on common bile duct ligation(BDL)-induced liver fibrosis in rats by regulating intestinal flora.Methods The 25 SD rats were randomly divided into five groups:the Sham group,the BDL group,the negative control adenovirus(NC adv)group,the miR-199a-5p adv group and the miR-199a-5p sponge adv group.The pathological changes of liver tissue and the degree of liver fibrosis were ob-served by HE,Masson and Sirius Red staining.The levels of aspartate aminotransferase(AST),alanine amin-otransferase(ALT),total bilirubin(TBIL)and direct bilirubin(DBIL)in serum of rats were determined by a fully automatic biochemical analyzer.The mRNA expression level of miR-199a-5p in liver tissue of rats was detec-ted by qRT-PCR.The protein expression levels of α-smooth muscle actin(α-SMA)and collagen type 1 alpha 1(COL1A1)in liver tissue of rats were detected by double immunofluorescence staining and Western blot experi-ment.Rat feces were collected for 16S rRNA high-throughput sequencing.Results The expression of miR-199a-5p was up-regulated in the liver tissue of BDL rats(P＜0.01).Compared with the NC adv group,the degree of liver injury and collagen deposition were relatively serious,the levels of AST,ALT,TBIL and DBIL in serum and the expression levels of α-SMA and COL1A1 in liver tissue increased in the miR-199a-5p adv group(all P＜0.05).However,the results of miR-199a-5p sponge adv intervention were opposite(all P＜0.05).The 16S rRNA sequencing results showed that rats treated with miR-199a-5p adv were characterized by increased diversity and richness of intestinal microbiota,changed composition of intestinal microbiota,while the results of miR-199a-5p sponge adv interfering with the bacterial community were opposite(all P＜0.05).Conclusion miR-199a-5p promotes liver fibrosis of BDL rats,and its mechanism may be related to regulating the diversity and abundance of intestinal microbiota.

Objective To investigate the effect of CXXC finger protein 4 (CXXC4) on the proliferation and apoptosis of pancreatic cancer PANC-1 cells.Methods The expression level of CXXC4 in pancreatic canc-er tissues and its relationship with prognosis and clinicopathological stage of the patients were analyzed in on-line databases.The qRT-PCR technique was used to detect the mRNA expression level of CXXC4 in human normal pancreatic ductal epithelial cell line (HPNE) and pancreatic cancer cell PANC-1,AsPC-1 and BxPC-3. si-NC,si-CXXC4,pcDNA3.1 and pCDNA3.1-CXXC4 were respectively transfected into PANC-1 cells.West-ern blot was conducted to detect the effectiveness of CXXC4 knockdown and overexpression.CCK-8,colony formation,EdU and immunofluorescence assays were conducted to analyze the effect of CXXC4 knockdown or overexpression on the proliferation and apoptosis of PANC-1 cells.The bioinformatic websites was used to predict the upstream microRNA (miRNA) of CXXC4.The Starbase database was adopted to analyze the cor-relation between miR-450b-5p and CXXC4 expression in pancreatic cancer tissues.Results The TCGA data-base results showed that the expression of CXXC4 in pancreatic cancer tissues was lowly expressed compared with in paracancerous pancreatic tissues (P<0.001),moreover which was associated with the overall survival and poor prognosis in the patients with pancreatic cancer (P<0.05).The GEPIA database analysis results showed that compared with stage Ⅰ pancreatic cancer,the CXXC4 expression in stage Ⅱ pancreatic cancer was decreased (P<0.05).Compared with HPNE cells,the CXXC4 expression in 3 kinds of pancreatic cancer cells was decreased (P<0.05).Compared with the si-NC group,the proliferation and colony formation ability of PANC-1 cells in the si-CXXC4 group were enhanced,the expressions of proliferation markers Ki67 and PC-NA were increased,and the expressions of apoptosis markers Bax,caspase-3 and caspase-9 were decreased;compared with the pcDNA3.1 group,the PANC-1 cells in the pcDNA3.1-CXXC4 group obtained the opposite results (all P<0.05).The bioinformatic websites predicted that miR-450b-5p was the upstream miRNA of CXXC4,CXXC4 in pancreatic cancer tissues was negatively correlated with the miR-450b-5p expression (r=-0.227) and miR-450b-5p in various mammalian species was highly conserved.Conclusion CXXC4 inhibits the proliferation of PANC-1 cells in pancreatic cancer and promotes theirs apoptosis.

Bispecific antibody (BsAb) has two different antigen-binding sites, divided into the "IgG-like" format and the "non-IgG-like" format. Different formats have different characteristics and applications. BsAb has higher sensitivity and specificity than conventional antibodies, with special functions such as recruitment of immune cells and blocking of dual signaling pathways, playing an important role in immune-diagnosis and therapy. With the deterioration of the global environment and the irregular living habits of people, the incidence of tumor is becoming higher and higher. Tumor becomes the most serious fatal disease threatening human health after cardiovascular disease. There are 12 million estimated new tumor cases each year worldwide. The major clinical treatments of tumor are surgical resection, chemoradiotherapy, target therapy. Tumor immunotherapy is a novel approach for tumor treatment in recent years, and activates human immune system to control and kill tumor cells. Although the traditional monoclonal antibodies have already acquired some therapeutic effects in tumor targeted therapy and immunotherapy, they induce drug resistance resulted from the heterogeneity and plasticity of tumors. Binding to two target antigens at the same time, BsAb has been used in the clinical treatment of tumors and obtained promising outcomes. This review elaborates the research progress and applications of bispecific antibody in clinical tumor therapy.
Antibodies, Bispecific/therapeutic use* ; Antibodies, Monoclonal/therapeutic use* ; Humans ; Immunotherapy ; Neoplasms/therapy*