腺病毒介导的白细胞介素10基因转移对胰岛β细胞凋亡及胰岛素释放的影响

Objective To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes mellitus. Methods The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose ( 16. 7mmol/L) for a 1 -hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant After induction of IL-1β, the levels of nitric oxide (NO) and nitric oxide synthase ( NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry. Results Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-lβ [ NO level ( nmol/106 cells): 52. 9 ± 3. 2 vs 227. 3 ± 26. 4, 235. 1 ±28.6, both P＜0.05; NOS level (U/106 cells): 9. 3 ±1.2 vs 29. 8 ±2.5, 30.5 ±2.8, both P ＜ 0. 05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1β. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24. 6% ± 1.0% vs 33. 3% ± 5. 1% , 32. 6% ±1.1%) (P＜0.05).The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4%±1.1% vs 19.2%±2.2%,20.6%±2.3%,P＜0.05).Conclusions IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function,protecting islet cells from IL-1β-mediated apoptosis and promoting islet cells survival.It is a potential therapy for type 1 diabetes mellitus.     

腺病毒介导的白细胞介素10基因转移对胰岛β细胞凋亡及胰岛素释放的影响

Objective To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes mellitus. Methods The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose ( 16. 7mmol/L) for a 1 -hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant After induction of IL-1β, the levels of nitric oxide (NO) and nitric oxide synthase ( NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry. Results Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-lβ [ NO level ( nmol/106 cells): 52. 9 ± 3. 2 vs 227. 3 ± 26. 4, 235. 1 ±28.6, both P＜0.05; NOS level (U/106 cells): 9. 3 ±1.2 vs 29. 8 ±2.5, 30.5 ±2.8, both P ＜ 0. 05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1β. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24. 6% ± 1.0% vs 33. 3% ± 5. 1% , 32. 6% ±1.1%) (P＜0.05).The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4%±1.1% vs 19.2%±2.2%,20.6%±2.3%,P＜0.05).Conclusions IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function,protecting islet cells from IL-1β-mediated apoptosis and promoting islet cells survival.It is a potential therapy for type 1 diabetes mellitus.     

腺病毒介导的干扰素-β基因对SMMC-7721细胞凋亡的影响

目的:观察腺病毒介导的干扰素-β(IFN-β)基因能否诱导人肝癌SMMC-7721细胞体外凋亡.方法:用HEK293A细胞扩增重组腺病毒介导的人干扰素-β(AdhlFN-β)基因,经纯化后用空斑形成实验法测定病毒滴度;分别用AdhIFN-β基因和重组腺病毒介导的绿色荧光蛋白(AdGFP)基因转染人肝癌SMMC-7721细胞.并设空白对照组.应用免疫细胞化学方法检测hIFN-β基因的表达,应用Hochest 33342荧光染色法和流式细胞术检测转染重组AdhIFN-β基因组、空载体组和对照组细胞的凋亡情况.结果:重组腺病毒经HEK293A细胞扩增、纯化后滴度可达2×1011 pfu/mL,且40 MOI的腺病毒即可获得95%以上的转染效率;免疫细胞化学法检测到外源性hIFN-β基因在SMMC-7721细胞中的蛋白表达产物;荧光染色法检测到重组AdhIFN-β基因组细胞明显发生了凋亡,而重组AdGFP组和空白对照组细胞凋亡不明显;流式细胞术检测到转染重组AdhIFN-β基因组细胞凋亡率[(28.27±4.21)%]高于转染空载体组[(2.08±0.89)%]和对照组[(1.53 4±0.70)%](F=377.625,P<0.001),后2组细胞凋亡率比较差异无统计学意义.结论:腺病毒介导的hIFN-β基因能诱导体外肝癌细胞发生凋亡.     

IL-1β诱导甲状腺细胞UGRP1表达及其与Fas/FasL介导凋亡的相关性

目的 探讨白细胞介素-1β(IL-1β)诱导甲状腺细胞子宫球蛋白相关蛋白1(UGRP1)的表达及其与Fas/Fas L介导细胞凋亡的相关性。方法 设立对照组、IL-1β组、IL-1β+抗Fas L抗体组,体外培养大鼠甲状腺细胞(FRTL-5细胞),采用Real-time PCR法检测各组细胞UGRP1及Fas mRNA表达水平;采用流式细胞术检测各组细胞凋亡率。结果与对照组比较,IL-1β组、IL-1β+抗Fas L抗体组UGRP1及Fas mRNA表达水平升高,差异有统计学意义(P <0.05);与对照组比较,IL-1β组甲状腺细胞早期凋亡率升高(7.49%±1.91%vs 28.46%±3.17%),差异有统计学意义(P <0.001),与IL-1β组比较,IL-1β+抗Fas L抗体组甲状腺细胞早期凋亡率降低(28.46%±3.17%vs 19.20%±1.75%),差异有统计学意义(P <0.05);与IL-1β组比较,IL-1β+抗Fas L抗体组UGRP1 mRNA (2.22±0.31 vs 2.66±0.28)及Fas mRNA(2.75±0.18 v...     

口服胰岛素抑制胰岛β细胞凋亡预防NOD鼠糖尿病

目的: 观察口服胰岛素免疫干预对非肥胖糖尿病(non-obese diabetic, NOD)鼠胰岛炎、β细胞凋亡和糖尿病的影响,并探讨其诱导免疫耐受的机制.方法: 86只NOD雌鼠随机分为胰岛素处理组(n=43)和磷酸盐缓冲液(phosphate buffered saline, PBS)对照组(n=43),从4周龄始每周灌胃人普通胰岛素1mg(70μL)2次,12周后改为每周灌胃1次至30周,对照组予等体积的PBS;于12周龄观察胰岛炎和胰岛β细胞凋亡;检测胰岛Fas和FasL的表达;测定血清IL-4和IFN-γ浓度,以及胰岛内I-Aβg7,IL-1β,IFN-γ,Fas,IL-4,TGF-β mRNA和小肠PP(Peyer's Patch)淋巴结IL-4,IFN-γ,TGF-β mRNA的表达水平.结果: NOD鼠口服胰岛素组30周龄和52周龄时发病率为55.6%和70.4%,分别比PBS对照组(85.7%和96.4%)低(P＜0.05).胰岛素组胰岛炎积分比对照组低,但差异无统计学意义(P＞0.05).胰岛素组胰岛Fas抗原表达和β细胞凋亡率均比PBS对照组低(均P＜0.05).胰岛素组胰岛内I-Aβg7,IFN-γ,IL-1β,Fas mRNA和PP淋巴结IFN-γ mRNA表达均较PBS对照组低(均P＜0.05),而IL-4,TGF-β mRNA表达较对照组高(均P＜0.05);胰岛素组血清IL-4比PBS组高,IFN-γ比PBS组低(均P＜0.05).结论:口服胰岛素能诱导NOD鼠的免疫耐受而预防糖尿病的发生,但不能阻断胰岛炎的进展.口服胰岛素能诱导调节性T细胞产生,使全身和胰岛局部T细胞由Th1向Th2转型,从而抑制Fas介导的β细胞凋亡而预防糖尿病.     

游离脂肪酸对人胰岛β细胞凋亡的影响及氨基胍的保护作用

目的:初步探讨游离脂肪酸对人胰岛β细胞凋亡的影响及其分子机制.方法:体外分离培养人胰岛细胞,免疫组化鉴定β细胞后分为对照组、游离脂肪酸组和氨基胍组,37℃、5%CO2培养72 h后做胰岛素释放实验,测定培养液上清胰岛素和一氧化氮(NO)水平.原位末端核苷酸标记法(TUNEL)和胰岛素免疫组化双染色法及ELISA检测胰岛β细胞凋亡,RT-PCR检测胰岛细胞p53 mRNA和bcl-2 mRNA表达水平.结果:游离脂肪酸组胰岛β细胞凋亡小体富计因子(6.37±3.4)、β细胞凋亡百分数(16.9%)、NO(187.35±20.99)μmol/L和p53 mRNA表达水平(0.645±0.029)均显著高于氨基胍组[分别为(1.17±0.2)、6.2%、(130.8±9.2)μmol/L和(0.47±0.02),P＜0.05]和对照组[分别为(1.11±0.4)、4.8%、(103.3±31.2)μmol/L和(0.35±0.03),P＜0.01],而胰岛素(9.27±1.13)mU·L-1·1×106cells和bcl-2 mRNA(0.131±0.046)表达水平则显著低于氨基胍组和对照组(P＜0.05).结论:游离脂肪酸可诱导人胰岛β细胞凋亡,其机制可能与胰岛β细胞抗氧化能力降低引起p53表达增加和bcl-2表达降低有关,氨基胍可部分防止游离脂肪酸诱导的人胰岛β细胞凋亡.     

血管紧张素Ⅱ对胰岛凋亡及Fas/Fas-L表达影响的实验研究

目的 观察血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)对体外培养犬胰岛细胞凋亡的诱导作用及氯沙坦(losartan)抑制胰岛凋亡和对凋亡相关基因Fas和Fas-L表达的影响.方法 将获得的纯化犬胰岛经不同处理培养48h后,采用光镜和透射电镜观察胰岛细胞凋亡情况,原位末端标记法检测胰岛细胞凋亡百分率,免疫组化法和流式细胞术检测胰岛细胞Fas和Fas-L表达.结果 体外培养犬胰岛经1.0μmol/L AngⅡ处理48h 后,胰岛细胞出现调亡(16.41±3.01)%现象,电镜见细胞皱缩、胞核裂解,电泳显示核酸断裂片呈"梯形"结构,Fas(11.46±1.97)%和Fas-L(14.68±3.51)%表达亦显著增强.氯沙坦组凋亡细胞数减少至(9.25±1.58)%,Fas(6.85±1.89)%和Fas-L(8.16±2.13)%表达减弱.两两比较差异有统计学意义(P<0.05).结论 AngⅡ诱导胰岛细胞凋亡.凋亡由AT1受体介导,Fas和Fas-L参与其凋亡过程.氯沙坦具有胰岛保护作用.     

完全弗氏佐剂抑制NOD鼠胰岛β细胞凋亡及其机制

目的:探讨完全弗氏佐剂( complete Freund’s adjuvant, CFA)对非肥胖糖尿病( nonobese dia-betic, NOD)鼠胰岛β细胞凋亡及凋亡相关基因Fas,FasL和Bcl-x表达的影响。方法:将4周龄NOD雌鼠随机分为CFA组(n=5)和生理盐水( NS)对照组(n=5 ) ,给CFA组鼠后脚板注射50μLCFA,对照者鼠后脚板注射等量NS。监测血糖,若血糖连续2 d≥11.1 mmol /L即诊断为糖尿病。当NOD鼠发生糖尿病或至30周龄时,处死动物,取胰腺组织制成薄切片,HE染色观察胰岛炎,采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记( TUNEL)及ABC免疫组织化学双标记染色观察并计数凋亡的胰岛β细胞,ABC免疫组织化学法染色观察并记数Fas,FasL和Bcl-x表达阳性细胞。结果:至NOD鼠30周龄时,CFA处理组鼠无1只发生糖尿病,对照组鼠有3只发生糖尿病;CFA处理组的胰岛炎积分低于NS对照组(1.820±0.962 vs. 3.020±1.040,P<0.05 ) ;CFA处理组胰岛β细胞凋亡率、Fas阳性细胞率、FasL阳性细胞率均低于NS对照组[ (10.2±2.8) % vs. (15.9±6.5) %,(54.9±14.5)% vs.(75.7±12.9) %,(20.3±10.4) % vs. (27.9±12.0) %,P<0.05] ,Bcl-x阳性细胞率高于NS对照组[ (74.9±10.7) % vs. (66.0±18.3) %,P<0.05]。结论:CFA能够减轻NOD鼠胰岛β细胞凋亡,其机制与减少促凋亡基因Fas和FasL表达及增加抑制凋亡基因Bcl-x表达有关。     

mIL-18腺病毒载体构建及在大鼠骨髓间充质干细胞中的表达

目的构建表达mIL-18基因的腺病毒载体,并转染大鼠骨髓间充质干细胞(rBMSCs),观察其在rBMSCs中的表达,为脑胶质瘤的基因治疗实验研究奠定基础。方法 mIL-18基因亚克隆于穿梭质粒pAdtrackCMV上,构建的pAdtrackCMV-mIL-18线性化后转化已含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞,挑选同源重组质粒,线性化后HEK293细胞包装,CsCI纯化。体外分离培养大鼠骨髓间充质干细胞,流式细胞仪检测细胞免疫表型;然后转染rBMSCs,通过荧光显微镜、RT-PCR、ELISA等方法检测mIL-18表达。结果成功构建了绿色荧光蛋白标记的腺病毒Ad-mIL-18,并在rBMSCs中高效表达。结论 pAdEasy-1是基因转染的高效系统,腺病毒Ad-mIL-18转染的rBMSCs可以作为脑肿瘤基因治疗研究的种子细胞。     

腺病毒载体介导的可调控鼠白细胞介素12基因对大肠癌小鼠的治疗效果

目的 构建携带鼠白细胞介素(mIL)12基因并可经RU486诱导表达的重组腺病毒载体,研究其对小鼠大肠癌移植瘤的治疗效果及安全性.方法 将穿梭质粒pDC-RUmIL-12与Admax腺病毒包装系统的辅助质粒pBHGloxΔE1,3Cre共转染HEK293细胞后,构建出可调控的复制缺陷性重组腺病毒载体,用mIL-12引物对病毒进行鉴定;氯化铯密度梯度离心纯化病毒,最终稀释法测定其滴度.体外感染结肠癌C26细胞后,用ELISA法检测其基因调控表达.皮下注射C26大肠癌细胞构建小鼠大肠癌动物模型,将60只小鼠分成4个治疗组(Ad-缓冲液对照组、Ad-RUmIL-12对照组、Ad-RUmIL-12+RU486治疗组和Ad-mIL-12治疗组), 从各治疗组的肿瘤大小、病理及血清丙氨酸氨基转移酶(ALT)水平等方面评价各组的治疗效果及不良反应.结果 经PCR鉴定证实可调控腺病毒载体Ad-RUmIL-12构建正确,纯化后病毒滴度达到4.62×1010空斑形成单位(pfu)/ml.病毒感染体外培养的C26细胞后,给予诱导剂RU486则最高可诱导表达mIL-12蛋白(516 ±43)pg/ml,而去除和不加入RU486时,只有少量的mIL-12蛋白表达[(38±3)pg/ml、(42±5)pg/ml].在小鼠动物模型上, Ad-RUmIL-12治疗组和Ad-缓冲液组肿瘤生长较快;Ad-RUmIL-12+RU486 治疗组和Ad-mIL-12治疗组对肿瘤生长有明显的抑制作用,病理检查显示两组肿瘤有较明显的坏死,而前组小鼠的血清ALT水平和不良反应发生率明显低于后组(4/15比10/15,P<0.05).结论 成功构建了可调控重组腺病毒 Ad-RUmIL-12,可以通过RU486诱导mIL-12蛋白的表达,并对大肠癌移植瘤有杀伤作用,明显提高了肿瘤基因治疗的安全性.     

Prevention of beta cell dysfunction and apoptosis by adenoviral gene transfer of rat insulin-like growth factor 1

Background Islet β-cells are almost completely destroyed when patients with type 1 diabete are diagnosed. To date, insulin substitute therapy is still one of the main treatments. The cure of type 1 diabetes requires β-cell regeneration from islet cell precursors and prevention of recurring autoimmunity. Therefore, β-cell regeneration and proliferation emerge as a new research focus on therapy for type 1 diabetes. Islet β-cell regeneration and development are controlled by many growth factors, especially insulin-like growth factor-1 （IGF-1）. Methods Recombinant adenovirus encoding rat IGF-1 （rlGF-1） was constructed and transduced into rat β-cells, RINm5F cells. Western blotting analysis and ELISA were used to detect rlGF-1 protein. Streptozotocin （STZ） was used to induce RINm5F cell destruction. The level of nitric oxide （NO） was detected in cell culture supernatants by the Griess reaction. Islet cell function was evaluated by glucose-stimulated insulin production. Flow cytometry analysis was further used to investigate the apoptosis of RINm5F cells. Thiaoollyl blue viability assay was applied to determine cell viability. Results The recombined adenovirus-rlGF-1 was successfully constructed and the titer was 4.0×10^8 pfu/ml. The rlGF-1 protein was effectively expressed in the RINm5F cells and cell culture supernatants, rlGF-1 expression remarkably inhibited STZ-induced islet cell apoptosis and significantly decreased the level of NO. Furthermore, IGF-1 expression also significantly protected insulin secretion and cell proliferation in a time-dependent manner. Conclusions Our study suggests that locally produced rlGF-I from RINm5F cells may be beneficial in maintaining β-cell function, protecting β-cells from the destruction of apoptosis factors and promoting β-cell survival and proliferation. IGF-1 might be considered as a candidate gene in gene therapy for type 1 diabetes. In addition, it appears that the apoptosis induced by STZ may be NO-dependent.     

幽门螺杆菌感染对冠心病患者内皮功能及血清同型半胱氨酸的影响

目的 观察感染幽门螺杆菌和未感染幽门螺杆菌的冠心病患者内皮功能及血清同型半胱氨酸(Hcy)水平的变化,探讨幽门螺杆菌感染和冠心病发病的关系.方法 应用ELISA法检测冠心病组患者(242例)和非冠心病组患者(88例)血清Hp抗体(HpIgG),比较两组HpIgG阳性率.应用放射免疫法检测冠心病组中HpIgG阳性和HpIgG阴性患者的前列环素I2(PGI2)、血栓素B2(TXB2)、内皮舒张因子一氧化氮(NO)、白介素-1β(IL-1β)、Hcy、叶酸、维生素B12(VitB12)水平.结果 冠心病组血清HpIgG阳性率明显高于非冠心病组HpIgG阳性率(53.3% 和 38.6%,χ2=5.556,P＜0.05).冠心病组HpIgG阳性者TXB2、6-K-PGF1α、Hcy水平分别为(262±86) pg/ml、(276±75) pg/ml、(18±6) μmol/L,HpIgG阴性者TXB2、6-K-PGF1α、Hcy水平分别为(216±65) pg/ml、(299±70)pg/ml、(16±5)μmol/L,两组上述指标水平间差别均有统计学意义(P＜0.05);冠心病组HpIgG阳性者和HpIgG阴性者NO、IL-1β、叶酸和VitB12水平间差别无统计学意义(P＞0.05).结论 幽门螺杆菌感染与冠心病发病有关,幽门螺杆菌可能导致内皮功能障碍及升高Hcy水平来促进冠心病的发生和发展.     

Expression of chemokine receptors CCR3,CCR5 and CXCR3 on CD4+ T cells in CBA/JxDBA/2 mouse model,selectively induced by IL-4 and IL-10,regulates the embryo resorption rate

Background Chemokines and their receptors have been a research focus in transplantation immunology.Chemokines and their receptors play a role in lymphocyte recruitment and differentiation process.This study aimed to observe whether IL-4 and IL-10 may regulate the expression of chemokine receptors CCR3,CCR5 and CXCR3 on CD4+ T cells in CBA/JxDBA/2 mouse model and to explore the role of CCR3,CCR5,CXCR3 in immune tolerance in pregnancy.Methods The mouse model of spontaneous abortion (CBA/JxDBA/2) and the normal pregnant mouse model (CBA/JxBALB/c) were used.CBA/JxDBA/2 mice were injected with IL-4 (CBA/JxDBA/2-1L-4),IL-4 and IL-10 (CBA/JxDBA/2-1L-4+IL-10),or normal saline (CBA/JxDBA/2-NS) as a control.The expression of CCR3,CCR5 and CXCR3 on CD4+ T cells from mouse peripheral blood was measured by the double-labelled FCM method,and the embryo resorption rate was also examined.Results The embryo resorption rate in the CBA/JxDBA/2 group without any treatment was significantly higher than that in the CBA/JxBALB/c group (17.9% vs 3.7%,P＜0.01).The embryo resorption rate in the CBA/JxDBA/2 group immunized with IL-4 or IL-4 together with IL-10 was significantly decreased,compared with that in the control and NS groups respectively.CCR3 expression on CD4+ T cells in the CBA/JxDBA/2 group without any treatment was significantly lower than that in the CBA/JxBALB/c group (0.3738±0.3575 vs 1.2190±0.2772,P＜0.01 );both CCR5 (3.0900±1.5603 vs 1.2390±0.6361,P ＜0.01)and CXCR3 (2.4715±0.9074 vs 0.9200±0.5585,P ＜0.01 ) expressions on CD4+ T cells of the CBA/JxDBA/2 group without any treatment were significantly higher than those of the CBA/JxBALB/c group.Significant up-regulation of CCR3 and down-regulation of CXCR3 were found in the CBA/JxDBA/2 group treated with IL-4 (CCR3:2.0360±0.6944,CXCR3:1.3510±0.5263,P ＜0.01) or IL-4 and IL-10 (CCR3:1.8160±1.0947,CXCR3:1.0940±0.7168,P＜0.01).Because of the CCR5,IL-4 and IL-10 (1.9400±0.8504 vs 3.0900±1.5603,P ＜0.05),but IL-4 alone (2.5310±1.3595 vs 3.0900±1.5603,P ＞0.05)treatment significantly decreased the expression of CCR5 in CBA/JxDBA/2.Conclusions The abnormal expression of CCR3,CCR5 and CXCR3 on CD4+ T cells may play an important role in the pathogenesis of spontaneous abortion.The pregnancy immune tolerance may be induced through selective induction of CCR3,CCR5 and CXCR3 expressions by IL-4 together with IL-10.     

阿魏酸钠对肾大部切除大鼠肾脏保护作用的研究

目的探讨阿魏酸钠对5/6肾切除大鼠肾脏病变的保护作用.方法大鼠随机分为4组,假手术组(Sham组),肾切除组(NX组),NX+阿魏酸钠组(SF组),NX+依那普利(E组).8周后检测大鼠血压、24h尿蛋白定量、血肌酐、BUN,肾皮质内皮素-1(ET-1)和一氧化氮(NO)等,观察肾组织病理改变,并应用RT-PCR的方法检测肾皮质内TGF-β1 mRNA的表达;Western蛋白印迹方法检测TGF-β1蛋白质的表达.结果术后大鼠出现明显的尿蛋白[(201.2±4.1)mg/24h,肾皮质ET-1含量显著升高,且有明显的肾小球硬化,而阿魏酸钠和依那普利均能减少尿蛋白[阿魏酸钠(78.1±3.1)mg/24h,依那普利(65.9±6.3)mg/24h,与假手术组比,P＜0.01],减少肾皮质ET-1含量和肾小球硬化指数.肾大部切除后肾皮质TGF-β1mRNA及其蛋白的表达明显升高,用药组上述指标明显降低(SF组和E组与NX组相比,P＜0.01).结论在5/6肾大部切除模型中,阿魏酸钠对5/6肾大部切除大鼠肾脏有明显的保护作用,其机制可能其减少肾脏ET-1的生成,抑制TGF-β1的表达有关.     

电针对坐骨神经分支选择性损伤大鼠痛觉过敏及脊髓NOS活性、NO水平的影响

目的 探讨电针对神经病理性痛大鼠痛觉过敏以及脊髓一氧化氮合酶(NOS)活性和一氧化氮(NO)水平的影响.方法 40只SD大鼠,随机分为空白组、假手术组、手术组和电针组(n=10).采用坐骨神经分支选择性损伤模型,电针"委中"和"环跳"穴,观察其对大鼠机械痛阈和热痛阈的影响,以分光光度法测定脊髓总NOS以及nNOS、iNOS活性和NO水平.结果 坐骨神经分支选择性损伤术可明显降低大鼠机械痛阈,手术组10d时为(14.83±0.79)g,与空白组和假手术组相比,差异有显著性(P＜0.05);并且手术组脊髓总NOS和nNOS活性以及NO水平均明显升高,分别为(44.23±7.81)U/mgprot和(39.64±10.28)U/mgprot以及(112.79±18.01)μmol/g,与空白组和假手术组相比,均差异有显著性(P＜0.01),iNOS却有降低的趋势;而电针干预后大鼠脊髓总NOS和nNOS活性明显降低,分别为(32.14±12.05)U/mgprot和(20.97±12.16)U/mgprot,NO合成也明显减少,为(70.96±18.15)μmoL/g,与手术组各项比较差异有显著性(P＜0.05,P＜0.01);与此同时显著减轻坐骨神经分支选择性损伤大鼠的机械痛敏状态,进而改善其痛行为.结论 电针减轻神经病理性痛可能与其抑制脊髓NOS活性、降低NO水平有关.     

CD40LIg基因修饰骨髓间充质干细胞对异种胰岛移植排斥反应的抑制作用

 目的 探讨T细胞分化群40补体免疫球蛋白（CD40LIg）基因修饰骨髓间充质干细胞（MSCs）对异种胰岛移植排斥反应的抑制作用。方法 建立Wistar-SD大鼠异种胰岛移植模型,用携带CD40LIg基因的重组腺病毒感染的MSCs进行干预治疗,观察糖尿病大鼠胰岛移植后生存情况、血糖变化和移植物病理形态学改变,检测移植物CD40LIg和胰岛素的表达以及移植大鼠白细胞介素2（IL-2）和肿瘤坏死因子（TNF-α）的水平变化。结果 (1)糖尿病大鼠血糖平均在移植后2天恢复正常,对照组血糖平均在移植后7天升高,单纯MSCs组和转染MSCs组血糖分别在20天和47天升高。(2)对照组、单纯MSCs组和转染MSCs组,移植物存活时间分别为（9±3.2）d、(25±5.3)d和(53±7.5) d,各组间比较具有显著差异(F =5.362, P < 0.05);移植糖尿病大鼠生存时间分别为（24±6.8）d、(51±7.9)d、(95±12.7)d,各组间比较差异具有显著性(F =6.821, P < 0.05)。(3)对照组在胰岛移植后7d内,IL-2和TNF-α的水平均急剧上升,显著高于移植前水平(P < 0.01)。(4) 两个治疗组移植物内均可见成片的胰岛细胞团,未见淋巴细胞浸润,转染MSCs组移植物内可见CD40LIg和胰岛素的表达。结论 CD40LIg基因修饰骨髓间充质干细胞可以延长胰岛移植物的存活时间,抑制大鼠异种胰岛移植的排斥反应。     

LPS介导三种啮齿动物原代胶质细胞炎症氧化反应的研究

目的 以Sprague-Dawley大鼠 (SD Rats) 、C57BL/6小鼠和昆明小鼠 (Kunming mice) 为研究对象, 探索研究脂多糖 (Lipopolysaccharide, LPS) 诱导3种鼠的原代混合胶质细胞释放炎症因子的对比.从而进一步探讨、发现研究三种鼠的应用价值、寻找更理想的炎症模型.方法 以一氧化氮合酶 (i NOS) 、环氧化酶 (COX-2) 、白介素-1beta (IL-1β) 和Nitric Oxide (NO) 作为代表激活的炎症氧化应激反应表征.获得C57BL/6小鼠、昆明种小鼠和SD大鼠23 d龄乳鼠后, 进行断头取脑和胶质细胞培养.传代至第3代接种于24孔板使用, 经LPS介导24 h后分别用Griess法检测NO浓度变化, Western b Lot检测COX-2、IL-1β、i NOS的蛋白表达.结果 用含N2的无血清培养基处理后的SD大鼠混合胶质细胞形态发生改变.比较三种鼠混合胶质细胞的正常对照组和LPS组, LPS组的NO释放量显著升高 (P<0.01) ;并且3种鼠混合胶质细胞的LPS组的i NOS/COX-2/IL-1β的表达也明显升高.结论 LPS可以诱导C57BL/6小鼠、昆明鼠以及SD大鼠混合胶质细胞NO的释放以及IL-1β、i NOS、COX-2蛋白的表达, 从而诱发炎症反应;LPS可以诱导3种鼠的原代混合胶质细胞形成炎症氧化模型.并且, SD大鼠反应较为灵敏.     

不同病程2型糖尿病患者胰岛α及β细胞功能评价

目的 了解不同病程2型糖尿病（T2DM）患者胰岛α、β细胞功能状况.方法 对283例T2DM患者（病程≤1年者40例,＞1年～≤5年者85例,＞5年～≤10年者90例,＞10年者68例）及30例正常对照者,行口服葡萄糖耐量试验（OGTT）及胰岛素、胰高糖素释放试验,比较各组间胰高糖素、胰高糖素/胰岛素比值等的变化,对胰高糖素行相关及回归性分析.结果 ①糖尿病组空腹及餐后各时相胰高糖素、胰高糖素/胰岛素比值及胰高糖素曲线下面积（AUC）,均高于正常对照者,且随病程延长各项指标相应升高,其中病程≤1年组0、30、60、120和180 min胰高糖素水平分别为[（71±20）、（106±36）、（143±54）、（133 ±68）和（87±55） ng/L],且随病程延长胰高糖素水平明显升高,病程＞10年组分别为[（80±19）、（125±36）、（167±47）、（178±64）和（129 ±65） ng/L].②不同病程T2DM患者间稳态模型胰岛素抵抗指数（HOMA-IR）及胰岛素敏感指数（ISI）差异无统计学意义,随病程延长,HOMA-β、糖负荷后30 min胰岛素和血糖净增值的比值（△I30/△G30）及胰岛素AUC均明显降低,差异有统计学意义（F值分别为3.75、3.77和3.07,均P＜0.05）,以△I30/△G30最明显.③多元逐步回归分析显示,胰高糖素与空腹血糖、血糖AUC呈正相关（t值分别为6.23、3.41,均P＜0.05）,与△I30/△G30呈负相关（t值为-2.13,P＜0.05）.结论 糖尿病早期在保护β细胞功能的同时应及早关注胰高糖素的调控,从而使血糖更易达标.