续断皂苷Ⅵ诱导的脂肪间充质干细胞复合纳米羟基磷灰石/壳聚糖支架治疗大鼠桡骨骨缺损

目的:探讨续断皂苷Ⅵ诱导的大鼠脂肪间充质干细胞(ADMSCs)联合纳米羟基磷灰石/壳聚糖(n-HA/CS)人工骨材料修复大鼠桡骨骨缺损的效果。方法:应用细胞表面抗原鉴定ADMSCs。取传至第3代的ADMSCs,培养基中加入续断皂苷Ⅵ诱导15 d。诱导后的ADMSCs和n-HA/CS复合培养获得的植骨材料修复大鼠桡骨骨缺损模型,于术后第4、12周骨缺损区行X光拍片并按照改良Gray标准进行结果分析。H-E染色,并按改良Nilsson的组织学评分标准进行成骨效应评估。结果:ADMSCs呈成纤维细胞样贴壁生长,高表达CD44。续断皂苷Ⅵ诱导后的细胞呈三角形、矮方形及多边形。茜素红染色可见散在的红色阳性结节,骨钙素表达阳性,阳性细胞率85.46%±5.22%。骨缺损修复术后4周移植材料已部分被吸收。术后12周实验组部分新生骨皮质恢复连续,开始出现骨髓腔。改良Gray评分显示,在各时间点实验组均高于各对照组,差异有统计学意义。第4周行H-E染色可见植入支架部分被吸收,实验组支架间隙和周围均充满细胞。第12周实验组出现类似骨单位样结构,骨基质中为淡粉色。改良Nilsson评分显示实验组在术后的4周和12周均高于各个对照组,有统计学意义。结论:续断皂苷Ⅵ能够诱导大鼠ADMSCs分化为成骨细胞,复合n-HA/CS支架能很好修复骨缺损。     

放射损伤对于移植的骨髓间充质干细胞在受体大鼠不同器官中植入的影响

目的： 探讨放射损伤对于骨髓间充质干细胞（MSCs）移植后在受体大鼠不同器官中植入的影响以及可能的机制。方法：采用DNA缺口末端标记法(TUNEL)检测正常对照组和单纯放射组大鼠心脏、肾脏、肝脏、肺脏的细胞凋亡率。应用密度梯度离心法结合贴壁法提取雄性大鼠骨髓MSCs，培养后把MSCs移植到［60COγ］照射和未经照射的雌性大鼠体内，通过PCR和Y染色体荧光原位杂交（Y- FISH）示踪雄性大鼠MSCs在雌性大鼠体内的分布情况。结果：照射后大鼠心脏、肾脏、肝脏、肺脏细胞凋亡率显著高于对照组。PCR结果显示照射后移植MSCs的大鼠心脏、肾脏、肝脏、肺脏和外周血中可以扩增出Sry基因的DNA序列，而且Y- FISH显示照射后移植MSCs的大鼠在心脏、肾脏、肝脏、肺脏中可发现Y染色体阳性的细胞。但是未经照射行MSCs移植的大鼠在上述器官中未发现Y染色体阳性的细胞。结论：全身放射促进组织细胞的凋亡，并导致移植的MSCs在受体大鼠心脏、肾脏、肝脏和肺脏的植入。     

肝硬化大鼠模型肝细胞移植途径的筛选

目的 通过比较4种同种异体肝细胞移植方法治疗肝硬化大鼠模型的疗效,筛选出较好的细胞移植途径.方法 50只经证实成模的肝硬化大鼠模型动物被完全随机分为5组:肝动脉移植组(G1)、门静脉移植组(G2)、脾动脉移植组(G3)、尾静脉移植组(G4)及对照组(G5).前4组分别自肝动脉、门静脉、脾动脉及尾静脉注射肝细胞悬液2 ml(细胞数量为2×106),对照组(G5)不作干预.移植前后对各组大鼠行肝功能检测及术后处死大鼠取肝脏作病理评分.结果 G1、G2组移植后前3周血清白蛋白水平逐渐下降,第5周后逐渐回升,到第7周达到峰值[(15.4±2.3)g/L,(15.4±2.1)g/L].G1、G2组移植后前3周血清总胆红素水平逐渐上升,到第5周后逐渐回落,到第7周达到移植前的水平[(13.62±2.32)μmol/L].G5组移植前后血清总胆红素水平变化不明显;术后病理G3组改善最为明显,G1、G2组次之,G4组再次之,G5改善情况最差.结论 在针对肝硬化大鼠模型不同的移植途径中,经脾动脉途径移植肝细胞,术后病死率较低,能更有效地改善肝脏功能及减轻肝细胞的变性和坏死.     

姜黄素对ERS诱导NASH大鼠肝细胞保护作用的研究

目的:探讨姜黄素是否通过抑制内质网应激(ERS)来减轻非酒精性脂肪性肝炎(NASH)大鼠肝细胞凋亡,从而发挥对肝脏的保护作用。方法:30只雄性SD大鼠随机分为正常对照组(n=10)、模型组(n=10)及姜黄素组(n=10)。模型组和姜黄素组采用高脂饮食喂养4周以建立NASH模型。继续喂养4周,姜黄素组每日给予姜黄素(200 mg/kg)灌胃,模型组与正常对照组给予等量生理盐水。4周后处死大鼠,留取血液及肝脏组织,检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平;HE染色观察肝组织病理学变化;Western blot法检测葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)蛋白的表达;TUNEL法检测细胞凋亡情况。结果:与正常对照组相比,模型组大鼠血清ALT和AST水平均显著升高,姜黄素组大鼠血清ALT和AST较模型组显著降低(P0.05);同时,姜黄素组大鼠的肝细胞脂肪变性及炎性程度均较模型组减轻,未见明显坏死灶。模型组GRP78和CHOP蛋白的表达水平均较正常对照组显著增加,姜黄素组GRP78和CHOP的表达水平较模型组显著降低(P0.01)。TUNEL结果表明,模型组大鼠肝组织内凋亡细胞较正常对照组增多,姜黄素组大鼠的凋亡肝细胞较模型组减少。结论:高脂饮食能诱发肝脏细胞发生过度ERS,从而启动细胞凋亡,导致NASH的发生。姜黄素减轻肝细胞凋亡的作用机制可能与其抑制ERS有关。     

大鼠胰岛素瘤实验模型的建立及其应用

目的 通过移植大鼠胰岛素瘤INS-1细胞至糖尿病小鼠肾包膜下使之形成胰岛素瘤,并诱导其发生凋亡,建立可用于研究胰岛β细胞凋亡机制的动物模型.方法 将5×106 INS-1细胞接种于链脲霉素(STZ)造模的糖尿病小鼠左肾包膜下,监测动物空腹血糖和血清胰岛素水平的变化,当血糖趋近正常后摘取动物左侧肾脏并检测其血糖和血清胰岛素水平的变化;固定包埋摘取的肾脏,进行HE染色以及胰岛素的免疫组化染色,确定胰岛素瘤模型的建立.在胰岛素瘤动物模型中,腹腔给予毒胡萝卜素(TG)或软脂酸钠(PA),监测给药后动物空腹血糖的变化,当血糖浓度出现逆转时,摘取动物左侧肾脏,通过TUNEL原位染色法检测移植瘤细胞的凋亡.结果 将INS-1细胞移植到糖尿病小鼠肾包膜下后,从第9天开始,动物空腹血糖进行性降低,血清胰岛素水平逐渐升高,当动物血糖接近至正常时,摘取动物左肾导致动物血糖显著升高,在摘取的左肾可见明显的移植瘤,免疫组织化学染色显示移植瘤细胞为胰岛素阳性.在胰岛素瘤动物模型给予TG或PA刺激后,动物空腹血糖出现逆转,显著升高,血清中胰岛素含量明显降低,摘取动物左侧肾脏后,TUNEL原位染色发现移植瘤内有明显的细胞凋亡.结论 大鼠胰岛素瘤INS-1细胞肾包膜下移植可以建立胰岛素瘤动物模型,应用此动物模型可以在体内研究胰岛β细胞凋亡的机制.     

锰诱导大鼠生精细胞凋亡过程中生殖激素和乳酸脱氢酶及PARP的作用

目的研究锰对大鼠体内生殖激素和乳酸脱氢酶(LDH)和多聚ADP核糖聚合酶(PARP)表达的影响,探讨它们在生精细胞凋亡过程中的作用。方法雄性SD大鼠48只,随机分为空白对照组,低剂量组和高剂量组,腹腔注射Mn Cl24周和6周,TUNEL法检测生精细胞凋亡,放射免疫测定血清睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)含量,化学比色测定血清和睾丸LDH含量。免疫组织化学法检测生精细胞PARP表达。结果各染锰组生精细胞凋亡指数、血清FSH、LH及LDH含量升高,血清T、睾丸LDH含量及PARP表达降低。结论锰可使大鼠T和睾丸LDH降低,FSH、LH和血清LDH活性升高,抑制大鼠生精细胞PARP表达,导致生精细胞凋亡。     

大黄酚对高脂饮食诱导的幼龄大鼠非酒精性脂肪肝的调节作用

目的探究大黄酚(chrysophanol,CP)对幼龄大鼠非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)的调节作用。方法将40只大鼠随机分为对照(control,Ctrl)组、CP组,高脂(high-fat,HF)组和HF+CP组。HF组和HF+CP组用高脂饲料喂养,其余2组以正常饲料喂养,CP组和HF+CP组同时腹腔注射CP(30 mg/kg)。4周后收集各组大鼠血清及肝脏组织,HE染色检测肝脏病理情况、TUNEL法检测细胞凋亡、Western blot检测脂质合成和分解代谢基因的表达、ELISA检测血清炎症因子水平。结果与Ctrl组比较,HF组大鼠肝脏组织出现明显的肝脂肪变性及炎性细胞浸润,凋亡小体明显增多;与HF组比较,HF+CP组大鼠肝组织脂肪变性及炎性细胞浸润显著减轻,凋亡小体数明显减少;同时,与Ctrl组比较,HF组大鼠脂质合成代谢基因ACC和SPEBP-1c的表达水平明显升高,分解代谢基因PPARα和CPT-1的表达水平明显降低,CP能显著减弱HP对ACC、SPEBP-1c、PPARα和CPT-1表达的影响。此外,HF能显著升高大鼠血清IL-1β和IL-6水平,CP能显著降低模型大鼠血清IL-1β和IL-6水平。结论 CP能通过调控脂质的合成代谢及炎症反应减轻幼龄大鼠NAFLD。     

糖尿病肾病大鼠血管紧张素Ⅱ1型受体自身抗体与肾脏损伤和细胞凋亡的关系

目的:探讨糖尿病肾病(DN)大鼠血管紧张素Ⅱ1型受体自身抗体(AT1-AA)与肾脏组织病理变化和肾小管上皮细胞及肾小球足细胞凋亡的关系。方法:36只雄性SD大鼠,随机分为正常对照组(NC组,n=6)和DN模型组(DN组,n=30),DN组大鼠给予高糖高脂喂养联合小剂量(35mg/kg)链脲佐菌素(STZ)腹腔注射建立DN模型。应用ELISA检测各组大鼠血清AT1-AA水平,并据此将各组再分为AT1-AA阳性和阴性亚组,检测各组大鼠血糖(BG)、肾功能指标及肾脏肥大指数(KW/BW);采用HE染色和透射电镜观察各组大鼠肾小管和肾小球病理变化;采用TUNEL法检测肾小管上皮细胞和肾小球足细胞凋亡。结果:DN组大鼠AT1-AA水平和阳性率明显高于NC组(P0.05或P0.01)。与NC组比较,AT1-AA阳性DN大鼠肌酐清除率(Ccr)显著降低,BG、肾功能指标及KW/BW明显升高(均P0.01);AT1-AA阴性DN大鼠除BG和KW/BW外,其它指标与AT1-AA阳性DN大鼠差异均有统计学意义(P0.05或P0.01)。与NC组比较,AT1-AA阳性DN大鼠均出现肾组织及超微结构病理变化,肾小管上皮细胞和肾小球足细胞凋亡率亦显著升高(P0.01);AT1-AA阴性DN大鼠肾脏未见明显病变,细胞凋亡率显著低于AT1-AA阳性DN大鼠(P0.05)。结论:AT1-AA水平变化与DN大鼠肾小管上皮细胞和肾小球足细胞凋亡有关,可能通过介导细胞凋亡而参与肾脏损害过程。     

白藜芦醇对糖尿病肾病大鼠肾脏的保护作用及可能机制

目的 观察白藜芦醇对糖尿病肾病大鼠肾脏的保护作用及能机制.方法 36只SD大鼠随机分为对照组(control组)、糖尿病肾病模型组(DN组)和白藜芦醇治疗组(Res组),每组12只,采用高脂饮食联合小剂量链脲佐菌素腹腔注射建立2型糖尿病大鼠模型.于造模成功后第8周,ELISA法检测各组大鼠尿液中24 h尿蛋白(24hTUP)水平以及肾组织中超过氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;生化分析仪检测各组大鼠血清肌酐(Scr)、尿素氮(BUN)、总蛋白(TP)、白蛋白丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)的含量;TUNEL染色观察肾组织细胞凋亡情况;Western blot检测肾组织中PI3K、Akt及p-Akt的表达.结果 给予白藜芦醇治疗后,糖尿病肾病大鼠血清中Scr、BUN及24 h TUP含量显著降低(P＜0.05);SOD活性升高,MDA含量降低(P＜0.05);肾脏细胞凋亡数量减少(P＜0.05);肾组织中PI3K与p-Akt的表达显著升高(P＜0.05),Akt的表达无显著差异(P＞0.05).结论 白藜芦醇能够减轻糖尿病大鼠肾功能损伤,抑制氧化应激反应,通过调控PI3K/Akt信号通路抑制细胞凋亡.     

三维多孔支架材料PLLA/n-HA的体外生物相容性研究

目的：研究三维多孔支架材料聚乳酸/纳米羟基磷灰石(PLLA/n-HA)的体外生物相容性,探讨其作为细胞培养材料和骨组织工程支架的可行性。方法将大鼠成骨细胞接种于PLLA/n-HA复合支架上,体外共同培养后,CCK-8法检测大鼠成骨细胞增殖活性,荧光倒置显微镜、扫描电子显微镜下观察PLLA/n-HA复合支架材料表面和孔隙内细胞粘附情况。结果 CCK-8法检测显示实验组复合支架材料上细胞的增殖与空白对照组的差异无统计学意义（P>0.05）;电镜观察到细胞在复合支架材料表面和孔隙内大量黏附、生长,并且随着共培养时间的增加,材料表面的细胞数量呈几何级增长。结论三维多孔支架材料PLLA/n-HA的生物相容性较好,可望成为一种性能良好的骨组织工程支架材料。     

A comparison of the biochemical properties of the human diaphorase (DIA3) isozymes determined by the common alleles DIA13, DIA23 and DIA33

(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.     

Mitomycin C-treated 3T3/B (3T3/A31) cell feeder layers in hybridoma technology

Several methods were compared for their efficiency at supporting the growth of hybridoma cells at low cell densities. Soluble growth factors from different sources gave poor results, whereas actively metabolising syngeneic cells proved to be effective feeder systems. Of the latter, the transformed subline of BALB/c embryo fibroblast 3T3/B cells - 3T3/A31 - were best. Macrophage (peritoneal exudate) feeder layers could be as effective as the 3T3/A31 cells, but were much more variable, probably reflecting the physiological state of the donor mice, the degree of sensitisation of the cells in vitro and the rate of recovery after isolation from the peritoneal cavity. Since the 3T3/A31 cells could replicate more rapidly than the hybridoma cells, division of the feeder cell chromosomes was inhibited using mitomycin C; 1-2 X 10(4) 3T3/A31 cells/ml were treated with 1 microgram/ml mitomycin C for 8-16 h at 37 degrees C, washed, and incubated for 3-7 days at 37 degrees C. The latter incubation was to permit the metabolism and breakdown of unreacted but intracytoplasmic drug, and the establishment of an active feeder layer before use with the hybridoma cells.     

HISTOCOMPATIBILITY DIFFERENCE BETWEEN C3HfeB/HeN AND C3H/HeN MICE: TUMOUR INDUCED IN C3HfeB/HeN MICE EXPRESSES C3H/HeN-ASSOCIATED ALLOANTIGEN

A transplacentally induced lung tumour of C3HfeB/HeN mouse origin expresses, as a tumour-associated antigen, a normal tissue component of strain A mice. The genetic locus coding for this alloantigen has been shown to be linked to the H-2 major histocompatibility complex. In the present study we demonstrate that this antigen is also expressed on normal tissues of C3H/HeN mice. Skin grafts exchanged between C3HfeB/HeN and C3H/HeN mice are reciprocally rejected at approximately 3 weeks after grafting. C3HfeB/HeN mice were derived from C3H/HeN mice in 1945. These strains have apparently deviated since then in their genetic regulation of the expression of the MHC-linked genetic locus. The finding of the C3H/HeN-associated antigen on a C3HfeB/HeN mouse-derived lung tumour indicates that this deviation is reversible.     

STAT3和SOCS3与子宫内膜异位症的相关性研究

目的: 探讨信号转导和转录活化因子3(STAT3)和细胞因子信号转导抑制因子3(SOCS3)的表达与子宫内膜异位症的相关性。方法: 采用Western blotting方法检测30例子宫内膜异位症患者异位(异位组)、在位子宫内膜(在位组)和25例正常子宫内膜(对照组)中总STAT3、磷酸化STAT3(p-STAT3)和SOCS3蛋白的表达情况。结果: 总STAT3蛋白在3组内膜中表达无差异,而其活化水平(p-STAT3/STAT3)在异位组最高,SOCS3蛋白表达在异位组最低,两者与在位和对照组相比较差异有统计学意义(P<0.05),相关性分析显示内异症患者中STAT3活化水平与SOCS3蛋白表达呈负相关(r=-0.499, P<0.01)。结论: 在子宫内膜异位症患者的异位子宫内膜组织中存在STAT3过度活化和SOCS3蛋白表达下降,两者呈负相关。     

Histocompatibility difference between C3HfeB/HeN and C3H/HeN mice: tumour induced in C3HfeB/HeN mice expresses C3H/HeN-associated alloantigen.

A transplacentally induced lung tumour of C3HfeB/HeN mouse origin expresses, as a tumour-associated antigen, a normal tissue component of strain A mice. The genetic locus coding for this alloantigen has been shown to be linked to the H-2 major histocompatibility complex. In the present study we demonstrate that this antigen is also expressed on normal tissues of C3H/HeN mice. Skin grafts exchanged between C3HfeB/HeN and C3H/HeN mice are reciprocally rejected at approximately 3 weeks after grafting. C3HfeB/HeN mice were derived from C3H/HeN mice in 1945. These strains have apparently deviated since then in their genetic regulation of the expression of the MHC-linked genetic locus. The finding of the C3H/HeN-associated antigen on a C3HfeB/HeN mouse-derived lung tumour indicates that this deviation is reversible.     

Adsorption of propylene and allene on TiCl3·1/3 AlCl3 and polymerization of allene with the system TiCl3·1/3 AlCl3/AlEt2Cl

Adsorption of propylene and allene from the gas phase on TiCl₃·1/3 AlCl₃ and TiCl₃·1/3 AlCl₃/AlEt₂Cl catalyst was measured volumetrically. In the ranges of 296–334 K and 1–100 kPa, the adsorbed amounts of both gases was found to be approximately proportional to the gas pressure. The adsorbed amount of allene is about twice as high as that of propylene under the same conditions. Low heats of adsorption of both propylene and allene support the assumption of the physical nature of the adsorption. Addition of AlEt₂Cl to the TiCl₃·1/3 AlCl₃ catalyst did not change substantially the sorption properties of the catalyst. Polymerization of allene with the system TiCl₃·1/3 AlCl₃/AlEt₂Cl in heptane was found to be first order in monomer and the rate decreases with increasing yield. A mechanism of retardation by the polymer formed is probably operative. Both heptane-soluble and heptane-insoluble polyallenes are produced. The soluble polymer has M?_n ≈ 10³ and a very narrow molecular weight distribution (M?_w/M?_n = 1,2 ± 0,2). Polyallene comprises mainly vinylidene units.