OBJECTIVE:To investigate the effects of calcium dobesilate on the ultrastructure of glomerular basement membrane(GBM)in diabetic rats.METHODS:30rats underwent unilateral nephrectomy,of whom,10were assigned to the normal control group(group NC),another20were induced to diabetic models(DM)by intraperitoneal injection of1%STZ,the models which have finished were divided into DM control group and calcium dobesilate group(group DD),each group admin?istered with distilled water and calcium dobesilate respectively,12weeks later,the change in the ultrastructure of kidneys and endogenous creatinine clearance rate(CrCl)in each group were observed.RESULTS:After12weeks,endogenous CrCl in group DD was significantly higher than that in group DM;Electron microscope showed that thickening of glomerular capillary basement membrane in group DD was less than that in group DM.CONCLUSION:Calcium dobesilate could improve the ul?trastructure in kidney of diabetic rats,prevent GBM thickening,and protect filtration barrier of renal glomerulus.

BACKGROUND:There is a linker between Hsp65 and Esat6 coding a segment of water repellent polypeptide.It is very gentle and easy to be folded,which is profitable to translate the keno-folding correctly between the two proteins and to make the space structure of fusional proteins consistent with those two native ones.Then,a correct structure is formed and the immunogenicity of the fusional proteins is improved.OBJECTIVE:This study was to clone the fusional genes Hsp65-Esat6 from mycobacterium tuberculosis(MTB)H37Rv, then to construct into a eukaryotic expressing vector which contains an enhanced green fluorescent protein(EGFP) reporter gene,and finally to identify the expression of Hsp65 protein and Esat6 protein by IHC methods.DESIGN:Single sample experiment.SETTING:Department of Microbiology ＆ Immunology,Medical College of Jinan University.MATERIALS:Plasmid pVAE was donated by Professor Cao from South China University of Technology.MTB H37Rv,E.coil DH5 α,expressing plasmid pEGFP-C1 and Hela cells were donated by Doctor Hu Ping.METHODS:The experiment was conducted at the Department of Microbiology ＆ Immunology and the Medical Experimental Center of Jinan University between September 2005 and June 2006.The whole-genome was extracted from MTB H37Rv by molecular cloning technique,and used it as template to amplify Hsp65 (no terminator) gene by polyrnerase chain reaction (PCR),to recombine it with pEGFP-C1 vector after purification to construct pEGHsp65(no terminator)recombination vector.pVAE vector was used as template to amplify Linker-Esat6 gene(with terminator)by PCR, and then to recombine it with pEGHSP65(no terminator)to construct an eukaryotic expressing vector pEGHsp65-Esat6 with the fusional genes Hsp65-Esat6 inside.Finally,genes Hsp65-Esat was checked by molecule biology methods such as PCR, restriction endonuclease and DNA sequencing.Hela cells were transfected with pEGHsp65-Esat6 and the expression of EGFP and the efficiency of transfection were observed.The expressions of Hsp65 protein and Esat6 protein were detected by IHC methods.MAIN OUTCOME MEASURES:①The size of pEGHsp65-Esat6.②The DNA sequencing result of the pEGFP-C1.③The expression of EGFP in the transfected Hela cells.④The IHC results of Hsp65 protein and Esat6 protein in Hela cells.RESULTS:①The size of pEGFP-C1 was 4.7 kb,that of pEGHsp65 was 6.4 kb,and that of pEGHsp65-Esat6 was 6.7 kb.There were differences between their speeds In agarose electrophoresis.②The results showed that it was the same as reported Hsp65 sequence and Esat6 sequence of MTB H37Rv.③After transfecting pEGHsp65-Esat6 for 24 hours,EGFP was found in 30% of Hela through Laser scanning confocal microscope. But there was no EGFP in non-transfected Hela.④Hsp65 protein and Esat6 protein with biological activities were detected in transfected Hela cells by IHC methods.CONCLUSION: Using Hsp65 and Esat6 as immunogens,we have successfully cloned and constructed a eukaryotic expressing vector which contain fusionaI genes Hsp65-Esat6 of MTB H37Rv and EGFP.

The effects of benazepril on P42/44MAPK, angiotensin Ⅱ expression in renal tissue and renal pathological change of the experimental diabetic rats were assessed and the possible mechanism of benazepril's renoprotective effect was explored. Adult male Wistar rats, 11-12 weeks age,weighing initially 160 to 200 g were randomly allocated into 2 groups: control group (A, n=6) and experimental group (n= 12). Diabetic rats in experimental group were rendered diabetic by intraperitoneal injection of Streptozotocin (60 mg/kg body weight), and randomly subdivided into B group (diabetic control) and C group (diabetic rats treated with benazepril, 6 mg/kg every day).Studies were performed 8 weeks after induction of diabetes. Twenty-four h urine of every rat was collected to detect urine creatinine. Serum glucose concentration and serum creatinine were determined by collecting blood samples from the inferior vena cava. Body and kidney weight were recorded. Creatinine clearance (Ccr) and ratio of kidney weight to body weight were calculated. Plasma and renal tissue angiotensin Ⅱ concentration was assayed by radioimmunoassay (RIA). The phospo-p44/42MAPK protein expression was detected by Western-blot. The results showed that benazepril had no significant effect on the blood glucose level in diabetic rats in two experimental groups.Ccr and ratio of kidney weight to body weight were increased in group B (P＜0. 01) as coapared with normal rats at the end of the 8th week. At the end of the 8th week, Ccr in group C was lower than that in group B (P＜0.01). The ratio of kidney weight to body weight in group C was lower than that in group B at the 8th week. There were glomeruli hypertrophy and slight or moderate mesangium proliferation in diabetic rats, while there was fragmentally proliferative mesangium in group C at the end of the 8th week. Renal tissue angiotensin Ⅱ concentration was significantly increased in group B, while benazepril could significantly decrease the concentration of angiotensin Ⅱ in renal tissue. The expression of the phospo-p44/42MAPK protein in group B was increased as compared with group A, while it was decreased in group C as compared with group B. P42/44MAPK pathway participated in the pathogenesis of diabetic nephropathy. Benazepril can eliminate high filtration of glomeruli, decrease proteinuria, and eliminate renal hypertrophy as well as renal destruction. Renoprotective effect of benazepril in diabetic rats may be partly related to the inhibition of angiotensin Ⅱ -P42/44MAPK pathway.

To observe the effects of calcium dobesilate on the expression of glomerular tissue inhibitor of metalloproteinase 1 (TIMP1), collagen Ⅳ, and ultrastructure of glomerular basement membrane in diabetic rats, rats model of diabetes was established by unilateral nephrectomy and intraperitoneal injection of 1% STZ (55 mg/kg), and rats were administered calcium dobesilate 100 mg/kg (DD group) or distilled water (DM group) respectively. 12 weeks later, the changes in the renal ultrastructure and creatinine clearance rate (Ccr) were examined in each group. The expression of glomerular TIMP1 and collagen Ⅳ were studied by immunohistochemical staining. Our results showed that after 12 weeks, the Ccr in DD group increased and was significantly higher than that in DM group. Electron microscopy showed that thickness of glomerular capillary basement membrane (GBM) in Group DD was less than that of DM group. No hyperplasia of collagen fibers was found, and the distance between the holes of endothelial cells in DD group was not as even as that in the normal group, but more even than that of DM group, and podocyte processes was still in order. Immunohistochemical staining of glomeruli showed that expression of TIMP1 and collagen Ⅳ in DD group were significantly less than those of DM group DM. It is concluded that calcium dobesilate can improve diabetic nephropathy by inhibiting the over- accumulation of collagen Ⅳ and calcium dobesilate may also contribute to diabetes by inhibiting the expression of TIMP1.

Objective:To explore the therapeutic strategy and result of adult total anomalous pulmonary venous connection(TAPVC).Methods:From November 2011 to November 2019, 6 adult patients with TAPVC underwent surgical correction. The Darling types include 4 cases of supracardiac , 1 case of intracardiac and 1 case of mixed type. There were 1 male and 5 female. The mean age was(28.6±4.8) years old and the mean weight was(47.3±3.67) kg. Preoperative oxygen saturation was 0.91±0.05.Results:All patients underwent primary repair successfully without perioperative death and complications. The average cardiopulmonary bypass time was(122.0±35.9) min, and the aortic cross-clamp time was(78.2±20.4) min. The mean postoperative hospitalization was(9.7±2.9) days, and the mean intensive care unit time was(3.5±1.4) days.The mean mechanical ventilation was(17.1±2.9) h. There were no later left heart dysfunction and pulmonary vein obstruction during the follow-up of 6-100 months.no pulmonary artery hypertension was identifed.Conclusion:TAPVC can be repaired savely in adult and satisfied result can be anticipated.

Objective:Analyze the effect of intracardiac method and upturning method in the treatment of infracardiac total anomalous pulmonary venous connection(TAPVC), to explore the surgical method of infracardiac TAPVC.Methods:From July 2011 to August 2019, 20 patients with infracardiac TAPVC were treated, including 12 cases with upturning method and 8 cases with intracardiac method. The cardiopulmonary bypass time, aortic cross-clamp time, delayed thoracic closure, ICU time, mechanical ventilation time, postoperative days and anastomotic flow rate were compared between the two groups.Results:There was no significant difference in cardiopulmonary bypass time, aortic cross-clamp time, delayed thoracic closure, ICU time and mechanical ventilation time between the two groups. The postoperative hospital stay in upturning group was significantly lower than that in intracardiac group [(14.7±2.9)days vs.(16.1±6.2)days, P<0.05], and the postoperative anastomotic velocity > 120 cm/s in intracardiac group was significantly less than that in upturning group(1 case vs. 7 cases, P<0.05). Two patients died in upturning group, but there was no significant difference compared with the intracardiac group. Conclusion:There is no significant difference between the two methods in the treatment of subcardiac TAPVC. The authors think that the exposure of the upturning methods is difficult, and the distortion of the anastomosis may be hidden trouble. The in situ anastomosis of the intracardiac method is not easy to make mistakes.

Objective:To evaluate the clinical value of lymph node dissection (LND) for intrahepatic cholangiocarcinoma (ICC) after surgical resection.Methods:A retrospective study was conducted on the clinical data of 156 patients who underwent surgery for ICC in Eastern Hepatobiliary Surgery Hospital of Naval Military Medical University from November 2010 to December 2017, including 94 males and 62 females, aged (60.0±9.5) years. Curative surgery was performed in 114 cases. Of 64 cases were in stage Ⅰ according to American Joint Committee on Cancer (AJCC), including 38 cases of non-lymph node dissection (NLND) and 26 cases of LND; 21 cases were in AJCC stage Ⅱ, including 11 cases of NLND and 10 cases of LND; 22 cases were in AJCC stage Ⅲb, including 14 cases of LND and 8 cases of lymph node resection (LNR); 5 cases were in AJCC stage Ⅲa, 2 cases were in AJCC stage Ⅳ. Univariate and multivariate Cox regression analysis were used for the risk factors of ICC prognosis. The log-rank test compared the survival rates of the two groups.Results:Cox multivariate analysis indicated that lymph node metastasis was independent risk factors for prognosis in patients with ICC ( HR=1.96, 95% CI: 1.09-3.55, P=0.026). A total of 114 patients were included in the curative surgery group. The 1-, 3-, and 5-year overall survival (OS) rates of the negative lymph node group ( n=91) were 65.9%, 47.3% and 35.6%, respectively, which were significantly better than those of the positive lymph node group ( n=23) who had 1-, 3-, 5-year OS rates of 56.5%, 17.7% and 0, respectively (χ 2=8.11, P=0.004 ). In stage Ⅰ and Ⅱ patients, there were no significant differences in 1-, 3-, 5-year OS rates between the NLND group and the LND group (both P>0.05 ). In stage Ⅲb patients, the LND group had 1-, 3-, 5-year OS rates of 71.4%, 29.8% and 0, respectively, significantly better than those of the LNR group who had 1-, 3-, 5-year OS rates of 37.5%, 0 and 0, respectively (χ 2=6.45, P=0.011). Conclusions:Lymph node metastasis is an independent risk factor affecting the prognosis of ICC. Lymph node dissection should be performed cautiously in ICC with AJCC stage Ⅰ and Ⅱ, while routine lymph node dissection is recommended in ICC with AJCC stage Ⅲb.

Objective:To summarize the experience of surgical treatment of anomalous aortic origin of the coronary artery arising from the inappropriate sinus(AAOCA) in children.Methods:Between April 2016 and November 2019, the clinical data of 9 patients with AAOCA in Anzhen Hospital were retrospectively analyzed, including 5 males and 4 females; aged from 5 month to 15 years old, with an average(11.7±5.1) years old. The clinical symptoms, auxiliary examination, treatment methods and prognosis of AAOCA patients were analyzed retrospectively.Results:The diagnosis of AAOCA was confirmed by echocardiography and coronary computed tomography angiography in all 9 cases. Left coronary arteries originated from the right coronary sinus in 4 cases, and right coronary arteries originated from the left coronary sinus in 5 cases. Two cases had a history of sudden cardiac death, and 1 case had a history of acute myocardial infarction; except for one asymptomatic case, other children had chest tightness, chest pain, syncope and other symptoms. Surgery was successfully completed in all children, included 7 cases of unroofing surgery, 1 case of coronary translocation and pulmonary translocation, and 1 case of inter-aortic release. There were no death in-hospital and serious complications. The postoperative follow-up period ranged from 3 months to 4 years. During the follow-up period, no patient died with normal heart function; 2 cases had nonspecific chest tightness and chest pain, and there was no evidence of myocardial ischemia.Conclusion:Younger AAOCA patients have a high rate of sudden cardiac death. Once diagnosed, early surgical treatment is needed. Coronary unroofing procedures and coronary translocation are recommended for AAOCA children with safe and reliable.