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Objective:To investigate the expression and significance of human ether-a-go-go related gene (HERG) protein in interstitial cells of Cajal (ICC) in patients with gallbladder stones.Methods:The gallbladder tissues of 60 patients with gallbladder diseases who underwent cholecystectomy from January 2018 to December 2020 in the Faculty of Hepato-Pancreato-Biliary Surgery, Chinese PLA General Hospital were collected, including 36 males and 24 females, aged (46.0±14.0) years. They were divided into two groups according to whether there were gallstones: gallstone group and control group (patients with gallbladder polyps and gallbladder adenomyosis), with 30 cases in each group. Color ultrasound was used to detect and calculate the gallbladder contraction rate. The neck, body and bottom tissues of the gallbladder were excised and sectioned. The expression of HERG protein and CD117 ( marker of ICC) was detected by immunofluorescence staining, immunohistochemistry and Western blot.Results:The gallbladder contraction rate in the gallstone group was (65.8±4.1)%, lower than that in the control group (73.8±5.3)%, with a statistically significant difference ( t=4.14, P<0.001). Immunohistochemistry showed that HERG protein was mainly distributed in the mucosal layer of gallbladder tissue, which was pale brown. The relative expression of HERG protein at the bottom of gallbladder in the gallstone group was (0.293±0.102), lower than that in the control group (0.694±0.059), with a statistically significant difference ( t=3.38, P=0.027). Immunofluorescence staining showed that HERG protein was mainly distributed in ICC of gallbladder epithelium. HERG protein expression in ICC at the bottom of gallbladder in gallstone group was lower than that in control group, while HERG protein expression at the neck and body of gallbladder had no significant difference. Conclusion:There are ICC and HERG protein in gallbladder tissue of patients with gallstone. The decrease of gallbladder contraction rate may be related to the decrease of HERG protein expression in ICC in gallbladder bottom tissue.
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Objective@#To evaluate the feasibility and strategy of the lesser omentum approach for laparoscopic pancreatic enucleation.@*Methods@#Between June 2011 and October 2013, 6 laparoscopic pancreatic enucleations were performed by lesser omentum approach.The average age was 42 years, including 1 male and 5 female.The preoperation diagnosis of 6 cases were pancreatic islet cell tumors determined by abdominal CT/MRI, ultrasound and digital subtraction angiography.The tumors of 3 cases located in pancreatic neck, 2 tumors located in neck and body of pancreas, and 1 tumor located in pancreatic body.Their biggest tumor diameter were 0.8-2.5 cm.@*Results@#Among the 6 cases, all laparoscopic pancreatic enucleations were successfully performed.None of the patients were converted to open operation.Eestimated blood loss was (26.7±18.6)ml, operating time was (82.5±19.4)minutes, and postoperative length of stay was (5.17±1.17)days.Additionally, postoperative complication included grade A pancreatic fistula in 1 case.After 36-64 months followed-up, there was no tumor recurrence and clinical symptom disappeared.@*Conclusion@#For the islet cell tumors located in pancreatic neck and body, the lesser omentumapproach may contribute to good surgical view and operative space, which can make pancreatectomy safer and easier for clinical application.
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Objective To investigate the clinical efficacy of colonoscopic metal stenting and ileus tube catheteriza-tion on left colorectal cancer with acute obstruction. Methods Clinical data of 80 patients meeting the diagnostic criteria for acute obstruction of the left half of colorectal cancer were included in this study. Patients were randomly divided into met-al stent group (n=40) and ileus tube group (n=40). The metal stent group was treated by metal stent and the ileus tube group was treated by ileus tube. Both were carried on by colonoscopy and X-line. After the relief of obstruction ( 7-10 d), patients were underwent colorectal cancer radical resection and anastomosis. The technical operations, improvement of obstruction and the efficacy of surgical treatment were compared between two groups. Results The success rate was 87.5%in metal stent group and the 97.5%in ileus tube group. There was no significant difference in the success rate between two groups (P>0.05). No complications were found in two groups. The operation time and the treatment cost were higher in metal stent group than those in ileus tube group. The relief rates of obstruction were 100%and 95%for metal stent group and ileus tube group, and there was no difference between them. Two cases were operated for emergency because of the failure of obstruc-tion relief. The relief time of obstruction and the difference between C-reactive protein values were much better in metal stent group than those of ileus tube group. After the obstruction relief, patients underwent a radical resection of the tumor and anastomosis, no anastomotic leakage was found in two groups. There were no significant differences in the operation time, in-cision infection and hospitalization time between two groups. Conclusion Anal ileus tube catheterization has a better eco-nomic value in the treatment of acute obstruction, but the efficacy of metal stent is better.
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Objective To investigate the mechanisms of the alleviation of islet graft earlier injury in the gastric submucosa.Methods The recipients were divided into gastric submucosa group (n =8) and hepatic portal vein group (n =8).1200IEQ SD rat islets were transplanted into diabetic SD rats induced by the administration of streptozocin (STZ).Glucose tolerance test and pathological examination were performed 14 days post transplantation.30 min after the transplantation,the C-peptide of the two groups were detected.12 h after the transplantation,IL-1β and TNF-α of the two groups were examined.Results The mean survival time (MST) of grafts in gastric submucosa group was (25.9 ± 4.1) d and (16.0 ± 0.8) d (P ＜0.01) in portal vein group.The results of glucose tolerance test and immunofluorescent staining demonstrated that grafts in gastric submucosa group survived well and got excellent function 14 days post transplantation.Compared with the gastric submucosa group,after 30 min of the transplantation,C-peptide in portal vein group was significantly higher [(1.46 ± 0.28) ng/ml.vs.(3.84 ± 0.22) ng/ml,P ＜ 0.01].Additionally,the level of IL-1β [(29 ± 1.41) pg/ml.vs.(262.26 ± 53.37) pg/ml,P ＜ 0.01] and TNF-α [(23 ± 1.41) pg/ml.vs.(138.51 ± 39.5) pg/ml,P ＜ 0.01] in portal vein group,were also significantly higher than the gastric submucosa group,after 12 h of the transplantation.Conclusion Islet transplantation in gastric submucosa prolongs islet grafts survival by avoiding or alleviating IBMIR and the injuries induced by early inflammatory mediators.
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According our practice of raical pancreaticoduodenectomy for pancretic head carcinoma and combined with these reviews, we suggested the active and palliative pancreaticoduodenectomy should be aviod. Skeletonization of hepatoduodenal ligament and the retroperitoneal resection should be the routine procedure in pancreticoduodenectomy, and at least invovle two regional lymph nodes. In addition, regardless of the metastase of No 13 lymph node, ristricted retroperitoneal resection for resectable pancretic carcinoma was needed. Exposured the superior mesenteric artery and distinguished inferior of uncinate process of pancrease with the artery, were the key point of the uncinate process of pancrease resection. Preoperative evaluation of angiography and other images, the ratio of activeness and combination with vessel resection would be improved. The style of pancreaticojejunostomy could be selected by the experience of the operator, we are apt to the double-deck invaginated pancreaticojejunostomy. Additionally, utilization of the electronic surgical workstation, should be careful and also need to accumulate more experience.
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Objective:To investigate RNA interference and apoptosis induced by LPS in kidney epithelial cells through silencing C5aR gene with small hairpin RNA (shRNA).Methods:We construct the eukaryotic expression vector of small hairpin RNA targeting rat C5aR gene,and transfected RK3E cell by electroporation,after G418 selection,so we got the stable cell line expressing C5aR shRNA.The experiment was designed into 3 groups:①normal control group,RK3E cells without transfection;②negative control group,RK3E cells transfected with blank vector;③ experimental group,RK3E cells transfected with C5aR shRNA.After incubation with LPS for 12 h,the ratio of apoptosis was tested by flow cytometry,the level of mRNA was tested by RT-PCR,and binding of ~(125)I-rrC5a to RK3E cells stimulated with LPS were performed to examine the expression of C5aR in RK3E cells.Results:Compared with the normal control group and negative control group,in the experimental group the ratio of apoptosis was significantly decreased(P＜0.01),and the expression of C5aR mRNA was significantly inhibited(P＜0.01),and binding of ~(125)I-rrC5a to RK3E cells was significantly decreased also.Conclusion:Hairpin shRNA targeting C5aR gene can lead to obvious gene silence in vitro and inhibit the cell apoptosis induced by LPS in kidney epithelial cell.
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Objective To investigate whether porcine Sertoli cells eould resist xenoreactive antibodies mediated complement lysis. Methods Sertoli cells were isolated from testes of 10 to 15 day-old landrace pigs. Α-Gal expression on Sertoli cells was measured by FACS and cytoimmunofluorescence. The binding of human se-rum IgG and IgM with Sertoli cells was assayed by FACS. After the incubation of the cultured Sertoli cells with 20% human B serum in vitro, the cellular lysis and cytotoxicity assay were detected with CytoTox-ONETM homogeneous membrane integrity assay and MTT method, and then activation of the complement cascade was examined by immunohistochemistry and immunofluorescence. The SV40-porcine endothelium cells line (SV40-PED) was served as control cells. Results α-Gal expression was found on Sertoli cells by FACS and cytoimmunofluorescence. After incubation with 20% human serum, cellular lysis ratio of the SV40-PED was 53. 13% ± 14.53%, while lysis ratio of the Sertoli cells was significantly lower (24.38% ±0.50%, P<0.01), the viability of Sertoli cells and SV40-PED were 98.73% ± 18.84% and 52.43% ± 8.08%, respectively. With immunohistochemistry and immunofluorescence, C3c and C4d were found binding on both the Sertoli cells and SV40-PED cells, however, C5b-9 was only detected on SV40-PED cells. Conclusion In vitro, compared with the SV40-PED, Sertoli cells could resist xenoreactive antibodies of human serum mediated complement lysis by preventing the C5b-9 formation.
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Objective To investigate the effects of the overexpression of HO-1 induced by CoPP in the donor on the survival of transplanted allogeneic islets of rats and the mechanism.Methods (1) Brown Norway rats were randomly divided into control group, and CoPP-induced group receiving intraperitoneal injection of CoPP (2.5 mg/kg) at 3rd and 1st day prior to islet isolation.By using the cytoimmunofluorescenee and Western blot, the expression of HO-1 in isolated islets was detected.The insulin level in the supernatant of the cultured islets stimulated with glucose was determined by ELISA.(2) Lewis male rat diabetic models were established by a single intravenous injection of alloxan, and then randomly divided into CoPP group and control group.Islets were transplanted under the left kidney capsule of each diabetic recipient.The survival time after transplantation, and pathological changes following rejection of the islet grafts were analyzed.Results The HO-1 was highly expressed in the islets isolated from CoPP-treated rats by cytoimmunofluorescence and Western blot.After stimulation with 16.7 mmol/L glucose, the insulin concentration in Copp-treated and Copp-untreated groups was (46.60± 1.13) and (19.01 ± 1.49) mIU/L respectively (P<0.05).The insulin concentration in Copp-treated and Copp-untreated groups in islets stimulated with 5.6 mmol/L glucose was (15.65 ± 0.89) and (12.28 ± 0.89) mU/L respectively (P>0.05).The stimulated index in Copp-treated and Copp-untreated groups was (2.98 ± 0.10) and 1.55 + 0.01 respectively (P< 0.05).The survival time of islets allograft in Copp-treated and Copp-untreated groups was separately (12.20±5.67) and (5.60± 1.14) days respectively (P<0.05).Histological analysis revealed the presence of more islands of insulin-positive cells and considerably fewer lymphocytes or inflammatory infiltration than the controls.Conclusions CoPP could induce the HO-1 expression of islets, and improve their function.Over-expression of HO-1 in islets could prolong survival time of islets allograft.
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Objective To investigate whether cotransplant with xenogenetic neonatal porcine Sertoli cells (NPSCs) could prolong rat islet allograft survival and its mechanisms.Methods 1500 islets equivalent quantity (IEQ) and 1×10~7 NPSCs were implanted under renal capsule of diabetic Wistar rats.Islets implanted alone were used as control group (n=6);islets co-transplanted with NPSCs under left renal capsule of recipients served as experimental group (n=6);meanwhile,islets and NPSCs implanted into the different sides of kidneys were used as another control grouP(n=4).Blood glucose level was measured everyday.The graft-bearing kidneys at the time of rejection were Results Co-transplantation with NPSCs to the same site significantly prolonged islet allograft survival (mean survive time,16.3±1.4 days vs.5.7±1.0 days in islet transplant alone control group,P<0.05).In contrast,transplantation with NPSCs and islets separately did not prolong the islet allograft survival (5.3±0.5 days).HE staining showed plenty of local infiltrated lymphocytes in the transplanted site of the eontrol group.which were demonstrated as mainly CD3+ T cells by immunopathology.The local expression of Bcl-2 was markedly elevated in co-transplantation group as compared with the other 2 groups,while there were no significant differences in the HO-1 expression among these groups.Conclusion Co-transplantation with xenogenic NPSCs can significantly prolong islet allograft survival in rats.The immunoprotective mechanism may be associateel with the inhibition of lymphocyte infiltration and the enhancement of the local expression of protective gene Bcl-2.
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Objective To investigate whether porcine endothelial cells transfected with SP1 de-coy ODNs could resist complement-mediated cytotoxicity during the model of SV-40-PED with human serum in vitro. Methods Immortalized porcine aortic endothelial cells of the line PED were divided in-to three groups. The transfected group was SV-40-PED with SP1 decoy ODNs. The mismatched group was SV-40-PED with scrambled SP1 decoy ODNs. The negative group was cells with oligo-fectamine only. The expression of α1,3-GT mRNA and αGal was detected after 26 h by using fluores-cence microscope, Western blot, RT-PCR and lactate dehydrogenase (LDH) activity assay. Results Fluorescence microscopy observed the decreased fluorescence of αGal after SP1 decoy ODNs transfec-tion. Dotted fluorescent decrease could be observed on some membrane while the mismatched group and negative group with bright green fluorescence. Western blot showed that the average absorbance of the PED cells transfected with decoy ODNs was decreased to 52.6% of the negative group (P<0.05). The expression of α1,3-GT mRNA in the PED cells transfected with decoy ODNs was 0.42±0.20 (isoform 1) and 0.27±0.12 (isoform 2) respectively, significantly lower than in the negative group (isoform 1, 0.72±0.17; isoform 2, 0.50±0.19; both P<0.05). The expression of α1, 3-G Tin the mismatch group was not different from that in the negative group (P>0.05). 20% normal hu-man serum (NHS) and 40 % NHS had cytotoxic effect on both mismatch and negative groups, but de-coy ODNs could confer SV-40-PED protection from the cytolysis effect (P<0.05), which made a re-markable reduction of complement-mediated cytotoxicity towards SV-40--PED. Conclusions SP1 decoy ODNs could confer porcine endothelial cells protection from complement-mediated cytotoxicity effect in vitro.
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Objective To establish the method for isolation and culture of porcine Sertoli cell sand detect the expression of the immune privilege-related molecules in the cultured cells. MethodsTestes were aseptically removed from the 10-to 15-days old Large White piglets. Testes were decap-sulated, minced and then digested with 0.2% (W/V) collagenase type V, 0.25% (W/V) trypsin and 0.05% (W/V) DNaseI. In the primary isolation, Sertoli cells were cultured at 37℃with 5% CO2.Inverted phase contrast microscopy and HE staining were used to observe the morphology of Sertoliceils, and the Sertoli cells were identified under an electron microscope. Viability and apoptosis ofcultured cells were measured with the AnnexinV-PI staining by flow cytometry. The expression of sox9, FasL, TGF-β and clusterin in Sertoli cells was detected by RT-PCR. The viability of long-termcultured Sertoli cells was assayed by MTT. Results In the cultured total cells, Sertoli cells accountedfor more than 90%. The apoptosis rate and mortality of Sertoli cells was (2.61±0.96)% and (2.12±0.74)% respectively. RT-PCR revealed that the expression of sox9, TGF-β and clusterin in theSertoli cells was strongly positive, but FasL was weakly positive. Viability of cultured cells measuredby MTT conformed that the sertloli cells could survive more than 21 days. Conclusion The isolationand culture methods of the neonatal porcine Sertoli cells was established. Under the culturedconditions, the Sertoli cells can express the immune privilege molecules and successfully survive at least 21 days.