抗霉素诱导肾小管上皮细胞铁死亡的实验研究

目的:观察抗霉素诱导肾小管上皮细胞发生铁死亡(ferroptosis)的现象,初步探讨铁死亡参与急性肾损伤的病理生理机制。方法:以1、2. 5、5、10μmol/L抗霉素A(antimycin A)干预人近曲小管上皮细胞(HK-2) 2、4、8、16、24h。MTT法检测细胞存活率,乳酸脱氢酶检测细胞活性,透射电镜检测细胞线粒体形态,Western blot检测凋亡关键蛋白(caspase3)活化,流式细胞术检测细胞内活性氧(ROS)。结果:HK-2细胞存活率随着抗霉素作用剂量和时间的增加而下降(P 0. 01)。2. 5μmol/L抗霉素A干预HK-2细胞2h后,细胞上清液乳酸脱氢酶(LDH)释放明显增加(P 0. 01),电镜观察发现铁死亡特征性改变,细胞线粒体膜密度增加,线粒体嵴减少,细胞内ROS含量明显增加(P 0. 05)。与抗霉素模型组相比,铁螯合剂去铁胺(deferoxamine)、铁死亡特异性拮抗剂Ferrostatin-1预处理HK-2细胞能明显减少抗霉素A损伤后LDH的释放、线粒体损伤及ROS的产生(P 0. 05),且该过程不伴有caspase3活化。结论:在缺氧诱导肾小管上皮细胞损伤过程中,铁死亡可能是肾小管上皮细胞较早出现的损伤方式;抑制细胞铁死亡的发生可以减轻缺氧对肾小管上皮细胞的损害。     

5-氮杂胞苷诱导大鼠骨髓间充质干细胞分化为心肌细胞的实验研究

目的 :观察 5 氮杂胞苷 （5 Azacytidine）能否诱导骨髓间充质干细胞分化为心肌样细胞及其分化率 ,并探明骨髓间充质干细胞分化为心肌样细胞外 ,尚有哪些细胞类型存在 ?方法 :以成年SD大鼠为研究对象 ,分离单个核细胞 ,用 5 氮杂胞苷诱导骨髓间充质干细胞的分化。通过倒置显微镜观察受到诱导的骨髓间充质干细胞形态的变化 ,用免疫组化的方法标测分化的心肌样细胞的肌钙蛋白I（troponinI）和结蛋白 （desmin）是否存在 ?并用放免试剂盒测定分化的心肌样细胞能否分泌心房钠尿肽 ,透射电镜可以观察到分化的心肌样细胞内是否有肌丝和肌丝团的存在 ,RT PCR方法可以测定分化的心肌样细胞是否有Nkx2 .5和troponinI的cDNA的表达 ?从不同的层面鉴定诱导分化的心肌样细胞是否具有心肌细胞的特性和功能 ?并用流式细胞仪测定分化的心肌样细胞占细胞总数的百分比。结果 :① 5 氮杂胞苷可以诱导骨髓间充质干细胞分化为心肌样细胞。②在骨髓间充质干细胞分化为心肌样细胞的过程中 ,有部分成纤维细胞生长和未分化的单个核细胞存在。结论 :5 氮杂胞苷可以诱导骨髓间充质干细胞分化为心肌样细胞 ,5 氮杂胞苷诱导骨髓间充质干细胞分化为心肌样细胞后再做细胞移植 ,可能有改善受损心脏的功能。     

骨髓间充质干细胞向心肌分化的实验研究

目的:研究骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)分化为心肌样细胞的能力,用于心肌补片治疗心肌梗死的研究。方法:分离C57/BSL小鼠BMSCs,全培养差速贴壁法,经过贴壁培养至第3代,流式细胞仪鉴定细胞表面标志(CD34、CD45、CD73、CD90),经10μmol/L的5-氮杂胞苷诱导细胞,24 h后更换完全培养基培养,2 w后进行免疫荧光染色,荧光显微镜观察心肌钙蛋白T(cTnT)和连接素蛋白43(CX43)的表达。结果:流式鉴定结果显示CD34、CD45阴性,CD73强阳性,CD90弱阳性。免疫荧光染色显示,诱导后细胞高表达心肌细胞特异性蛋白cTnT,连接素蛋白CX43表达水平明显增加。结论:5-aza可以诱导BMSCs大量表达心肌特异性蛋白cTnT和细胞连接素蛋白(CX43),干细胞分化为心肌样细胞,为干细胞移植治疗小鼠心梗提供种子细胞。     

乙型肝炎病毒复制调控元件对HBV DNA疫苗诱导的免疫应答

目的研究乙型肝炎病毒（HBV）复制调控元件增强子Ⅰ（ENHⅠ）及前S2（Pre-S2）抗原基因对HBV DNA疫苗诱导的免疫应答的影响。方法采用常规聚合酶链反应（PCR）从HBV adr亚型全基因DNA序列中分别扩增HBsAg、PreS2-HBsAg、HBsAg-ENHI和PreS2-HBsAg-ENHⅠ基因片段，重组到VR1012载体中，构建4种HBV DNA疫苗，转染HepG2细胞并免疫Balb／C小鼠。通过细胞免疫化学、酶联免疫分析（ELISA）、酶联免疫斑点试验（ELISPOT）等方法检测其在HepG2细胞内的表达及小鼠的体液及细胞免疫应答。结果转染的HepG2细胞表达相应的目的蛋白．ENHⅠ及Pre-S2抗原基因均可增强HBV DNA疫苗转染HepG2细胞表达HBsAg；免疫接种小鼠后第2周产生抗-HBs及HBsAg特异性细胞毒T淋巴细胞（CTL），Pre—S2抗原基因可增强HBV DNA疫苗免疫Balb／C小鼠诱导的抗-HBs及HBsAg特异性CTL的产生，ENHⅠ基因对免疫应答无影响。结论ENHI及Pre—s2抗原基因均可增强HBVDNA疫苗转染HepG2细胞表达HBsAg．Pre-S2抗原基因可增强HBVDNA疫苗免疫Balb／C小鼠引起的免疫应答。     

Hepatic immune tolerance induced by hepatic stellate cells

The liver, which is a metabolic organ, plays a pivotal role in tolerance induction. Hepatic stellate cells(Hp SCs), which are unique non-parenchymal cells, exert potent immunoregulatory activity during cotransplantation with allogeneic islets effectively protecting the islet allografts from rejection. Multiple mechanisms participate in the immune tolerance induced by Hp SCs, including the marked expansion of myeloid-derived suppressor cells(MDSCs), attenuation of effector T cell functions and augmentation of regulatory T cells. Hp SC conditioned MDSC-based immunotherapy has been conducted in mice with autoimmune disease and the results show that this technique may be promising. This article demonstrates how Hp SCs orchestrate both innate immunity and adaptive immunity to build a negative network that leads to immune tolerance.     

雌二醇纳米粒对大鼠免疫性肝纤维化模型的作用

目的 比较β-雌二醇纳米化前、后对猪血清诱导的肝纤维化动物模型肝脏纤维化的疗效及雌二醇抗肝纤维化的机制.方法 将S-D大鼠随机分为健康对照组、模型对照组、雌二醇治疗组、雌二醇纳米粒组和空白纳米粒组,每组10只,除健康对照组外,其余4组均腹腔注射猪血清(0.5 mL/次,2次/周).雌二醇治疗组、雌二醇纳米粒组和空白纳米粒组在第9周给予腹腔注射相应的干预药物,每周2次;在12周末处死大鼠.免疫组织化学法检测平滑肌肌动蛋白(α-SMA)的表达;比色法检测一氧化氮合酶(NOS)、一氧化氮(NO)、总抗氧化能力(T-AOC)和谷胱甘肽(GSH);RT-PCR检测肝组织Ⅰ、Ⅲ型胶原、结缔组织生长因子(CTGF)、转化生长因子-β1(TGF-β1)及基质金属酶(MMP-1)、组织金属蛋白酶抑制剂(TIMP-1)mRNA的表达.结果 肝纤维化动物模型肝脏组织的Ⅰ、Ⅲ型胶原、TGF-β1、CTGF和TIMP-1 mRNA的表达在雌二醇纳米粒治疗组和雌二醇治疗组均有明显减少,MMP-1 mRNA的表达明显增多,与模型对照组、空白纳米粒组比较,差异有统计学意义(P＜0.05);经治疗后雌二醇治疗组和雌二醇纳米粒组的T-AOC、NO、NOS、GSH活性均有提高,与模型对照组、空白纳米粒组比较,差异有统计学意义(P＜0.05);但雌二醇治疗组和雌二醇纳米粒组比较,差异无统计学意义(P＞0.05).结论 β-雌二醇通过抗氧化、降低Ⅰ、Ⅲ型胶原产生,抑制与肝纤维化有关的细胞因子TGF-β1及其下游信号CTGF的表达,促进MMP-1表达但抑制TIMP-1的表达等发挥其抗肝纤维化作用.纳米化β-雌二醇的抗大鼠免疫性肝纤维化作用比无纳米化β-雌二醇更强.     

蛙皮素致大鼠胰腺慢性纤维化模型的建立

目的胰腺纤维化是慢性胰腺炎的主要病理特征,由于缺乏重复性好的研究模型,胰腺纤维化的发生、发展过程尚不清楚。本研究采用CCK类似物蛙皮素反复诱致大鼠胰腺炎,旨在探讨胰腺纤维化发生的分子机制。方法SD大鼠腹腔注射蛙皮素50μg/kg,每周3次,分别于第2周、第4周和第8 周处死大鼠,采用HE染色和VG胶色观察不同时期胰腺病理组织学变化和结缔组织增生情况。同时,采用RT-PCR法检测胰腺Colα1(Ⅰ)mRNA的表达水平。结果造模第2周和第4周,胰腺间质中可见明显的炎性细胞浸润;第8周,“管状复合体”形成,间质纤维结缔组织大量增生并向小叶内伸展,胶原纤维交织成网状;而且随着造模时间的延长,Colα1(Ⅰ)基因的表达逐渐上调。结论蛙皮素多次大剂量腹腔注射致反复急性胰腺炎发作可以诱导大鼠产生胰腺纤维化,Colα1(Ⅰ)mRNA水平随造模时间延长逐渐升高,其机制可能与细胞信号分子介导的胰腺星状细胞活化而发生表型转化有关。     

Experimental study of cardiomyopathy induced by glucocorticoids

Abnormal ECG changes were found in some patients who were treated glucocorticoid during long term. In experimental animals, chronic administration of glucocorticoid resulted abnormal ST and T changes of ECG, increased duration of action potential and induced electron microscopical changes of mitochondria. No significant changes were found in serum and myocardial potassium content.     

蛙皮素致大鼠胰腺慢性纤维化模型的建立

目的胰腺纤维化是慢性胰腺炎的主要病理特征,由于缺乏重复性好的研究模型,胰腺纤维化的发生、发展过程尚不清楚.本研究采用CCK类似物蛙皮素反复诱致大鼠胰腺炎,旨在探讨胰腺纤维化发生的分子机制.方法 SD大鼠腹腔注射蛙皮素50 μg/kg,每周3次,分别于第2周、第4周和第8周处死大鼠,采用HE染色和VG胶色观察不同时期胰腺病理组织学变化和结缔组织增生情况.同时,采用RT-PCR法检测胰腺Colα1(Ⅰ)mRNA的表达水平.结果造模第2周和第4周,胰腺间质中可见明显的炎性细胞浸润;第8周,"管状复合体"形成,间质纤维结缔组织大量增生并向小叶内伸展,胶原纤维交织成网状;而且随着造模时间的延长,Colα1(Ⅰ)基因的表达逐渐上调.结论蛙皮素多次大剂量腹腔注射致反复急性胰腺炎发作可以诱导大鼠产生胰腺纤维化,Colα1(Ⅰ)mRNA水平随造模时间延长逐渐升高,其机制可能与细胞信号分子介导的胰腺星状细胞活化而发生表型转化有关.     

Induction of an immune response through interaction of helper cells with an immune complex bound to the surface of B cells.

Adoptive anti-trinitrophenyl (Tnp) responses were elicited from Tnp-hemocyanin (Tnp-KLH)-primed cells by challenge with an immune complex of KLH and a Tnp conjugate of the Fab fragment of rabbit anti-KLH. Removal of T cells by treatment with anti-Thy 1.2 (phi C3H) + complement abolished this effect. Tnp-KLH primed cells that had been incubated with a Tnp conjugate of pneumococcal polysaccharide type III (Tnp-SIII), and which were then unable to respond to Tnp-KLH, made an anti-Tnp response upon incubation with rabbit anti-Dnp when transferred together with cells primed with rabbit gamma globulin. Since Tnp-KLH was not added in these experiments, it would appear that the cells were triggered by immune complexes on their surfaces consisting of Tnp-SIII and rabbit anti-Dnp with the help of T cells primed with rabbit gamma globulin; the presence of free antigen was evidently not required. Therefore, antigen-antibody complexes, when bound to the surface of B cells, may mediate T-B cell cooperation in the humoral immune response.     

水红花子对小鼠免疫性肝损伤的影响

目的:观察人用口服剂量40倍的水红花子对免疫性肝损伤小鼠形态学、生理生化学方面的影响,为临床安全用药提供依据。方法:观察水红花子20g.kg-1.d-1对BCG/LPS致免疫性肝损伤模型小鼠肝脏病理变化及血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)活性,肝组织超氧化物歧化酶(SOD)、丙二醛(MDA)含量的影响。结果:水红花子组免疫性肝损伤小鼠肝脏病理损伤明显,血清ALT、AST均显著升高(P<0.01),肝组织SOD值明显下降(P<0.05),MDA明显升高(P<0.05)。结论:人用口服剂量40倍的水红花子可提高免疫性肝损伤小鼠的肝脏损伤程度,具有肝毒性,临床不可长期过量服用。     

茶碱对哮喘患者支气管肺泡灌洗液中肥大细胞激活的影响

目的 :研究哮喘患者支气管肺泡灌洗液 (BALF)中肥大细胞的功能特性。方法 :2 9名轻度哮喘患者参加了本项研究。BALF中的细胞经冲洗后 ,加入含抗IgE抗体、腺苷或腺苷 +茶碱、抗IgE抗体 +茶碱的LP4试管中进行体外激发 ,检测BALF肥大细胞中的组胺释放量。结果 :浓度为 3 0 0nmol L和 1μmol L的腺苷仅可刺激哮喘患者BALF中肥大细胞释放 3 %～ 5 %的组胺 ,1μmol L的腺苷与茶碱联合作用后可略降低腺苷的促进作用 ,抗IgE抗体浓度在 0 0 1%可促进哮喘患者BALF中肥大细胞释放2 3 6%的组胺 ,与茶碱联合作用能降低约 4%的组胺释放。结论 :哮喘患者BALF中的肥大细胞对腺苷的刺激反应较差 ,但对抗IgE抗体刺激的反应性明显增强 ,茶碱可能通过抑制哮喘中肥大细胞分泌介质而起治疗哮喘的作用     

Hypothesis for the pathogenesis of sodium aurothiomalate (Myocrisin) induced immune complex nephritis.

Renal complications associated with gold salt treatment in rheumatoid arthritis occur in fewer than 5% of treated patients. Recent investigations have shown that the renal lesion manifested clinically as membranous glomerulonephritis is caused by immune complexes. This paper presents a hypothesis for the mechanism by which gold causes this lesion: autoimmunization due to released tubular antigen(s). This hypothetical mechanism is strikingly similar to that responsible for autologous autoimmune nephrosis in the rat (Heymann's nephritis).     

Experimental study on the apoptosis of cervical cancer Hela cells induced by juglone through c-Jun N-terminal kinase/c-Jun pathway

Objective: To study the regulatory effect and molecular mechanism of juglone on apoptosis of cervical cancer Hela cells. Methods: Cervical cancer Hela cells were cultured and treated with different dosages of juglone(10, 20, and 40 μmol/L, respectively) and c-Jun N-terminal kinase(JNK) inhibitor SP600125(10, 20, and 40 μmol/L, respectively). Then cellular proliferative activity and the expression of JNK/c-Jun pathway molecule and apoptotic molecule in the cells were detected. Results: After 6, 12, 18 and 24 h of treatment, the value for proliferative activity of cells treated with juglone was significantly lower than that of control group(P0.05), and the anti-proliferative effect was more significant as the treatment period and juglone dosage increased(P0.05). The protein expressions of Bax, Cyt C, Fas, Fas L, Caspase-3, p-JNK and p-c-Jun in cells treated with juglone were significantly higher than those of control group(P0.05), and the protein expressions of Bax, Cyt C, Fas, Fas L, Caspase-3, p-JNK and p-c-Jun increased more remarkably as the juglone dosage increased(P0.05). In cells treated with 40 μmol/L juglone and SP600125, the protein expressions of Bax, Cyt C, Fas, Fas L and Caspase-3 were significantly lower than those of cells treated with 40 μmol/L juglone(P0.05), and the protein expressions of Bax, Cyt C, Fas, Fas L and Caspase-3 reduced more remarkably as the SP600125 dosage increased(P0.05). Conclusion: Juglone can increase the expression of apoptotic molecules in mitochondrial pathway and death receptor pathway by activating JNK/c-Jun pathway, thus inducing apoptosis of cervical cancer cells.     

抗人DR5单克隆抗体诱导肝癌细胞株SMMC-7721凋亡实验研究

目的探讨抗人DR5单克隆抗体对肝癌细胞株SMMC-7721致凋亡作用。方法常规培养肝癌细胞SMMC-7721,通过MTT法检测细胞抑制作用,流式细胞术定量分析凋亡细胞率。结果抗人DR5单抗能够诱导肝癌SMMC-7721细胞凋亡,在抗人DR5单抗浓度2μg/ml作用48h,对肝癌SMMC-7721细胞的杀伤率可达50.10%,增加抗人DR5单抗浓度细胞凋亡作用无明显增加。结论抗人DR5单抗能够诱导肝癌细胞SMMC-7721凋亡,以死亡受体为靶点的抗体制剂为肝癌治疗提供新途径。     

穿通支原体诱导膀胱肿瘤实验研究

目的建立穿通支原体(Mpe)诱发的膀胱肿瘤动物模型,进一步证实穿通支原体感染在哺乳动物体内诱导癌变的作用。方法40只ICR小鼠分免疫抑制和非免疫抑制组(每组20只)。免疫抑制组隔日腹腔注射环磷酰胺制成免疫抑制模型。穿通支原体菌液灌胃(12只)和尿道上行感染(16只),NS对照(12只),诱导小鼠膀胱肿瘤的发生,实验组在第4,8,18w分批宰杀,取小鼠膀胱组织进行微生物学和病理组织学检查。结果两种方式感染均使穿通支原体成功定植。尿道上行方式感染的小鼠移行上皮细胞癌变检出率高于灌胃方式感染;感染至18w后,上行感染免疫抑制组和正常组小鼠膀胱移行上皮细胞形态均发生了癌性恶变,且免疫抑制组细胞形态恶变程度为高。感染18w后的癌变检出率明显高于感染4w或8w后的小鼠。结论穿通支原体感染可诱导小鼠膀胱移行上皮细胞癌的发生,并且肿瘤的恶性程度与宿主的免疫状态有关。