人内皮抑制素在酵母细胞中的表达及其促成纤维细胞有丝分裂活性

利用酵母真核细胞表达系统表达出重组人内皮抑制素 (Endostatin) ,并对其促成纤维细胞有丝分裂活性进行研究。方法 :采用逆转录 PCR技术自人肝组织获得人内皮抑制素的cDNA ,将其克隆入真核表达载体pYEX4T 1,构建含人Endostatin的重组质粒 (pYEXEndo) ;经全自动序列分析仪测序确证后 ,将此重组质粒转化入酵母细胞 (DY15 0 )中进行诱导表达 ,表达产物经SDS PAGE分析及Westernblot鉴定。以MTT掺入法初步检测了重组人内皮抑制素对NIH3T3细胞的促有丝分裂活性。结果 :经CuSO4诱导的含pYEXEndo的酵母细胞表达出重组人GST Endostatin的融合蛋白 ,此蛋白在凝胶上表现为一约 45kD的阳性区带 ,在Westernblot实验中可被GST特异性多克隆抗体识别。重组人GST Endostatin融合蛋白的粗提物在体外能刺激NIH3T3细胞增殖。结论 :人GST Endostatin的融合蛋白在酵母表达系统中高水平表达 ,并具有刺激成纤维细胞生长的生物学活性。     

IL-7对胸腺T细胞及树突状细胞体外分化发育的影响

目的:探讨IL-7对胸腺T细胞及胸腺树突状细胞分化的影响。方法:摘取15～16日龄胎鼠胸腺进行体外器官培养(胚胎胸腺器官培养-FTOC),分别将细胞因子IL-7和培养基滴加在胸腺小叶上,12天后收集不同条件下经FTOC培养获得的胸腺细胞,流式细胞仪检测细胞表面分子CD4、CD8、CD11c、B220、Ia等的表达,通过光学显微镜观察细胞形态,通过细胞计数检测细胞数目的变化。再将经FTOC培养获得的胸腺细胞和异源的T细胞进行混合淋巴细胞反应,通过MTT法检测T细胞的增殖情况。结果:细胞计数结果表明添加外源性IL-7组的胸腺细胞数目明显减少,流式细胞仪检测结果显示其中胸腺CD4-CD8-双阴性细胞及CD8+单阳性细胞比例有所增加,而CD4+CD8+双阳性细胞比例显著下降,CD4+单阳性细胞比例没有明显变化;此外,B细胞和树突状细胞、NK细胞数量均有不同程度的增加。结论:IL-7在胸腺T细胞及胸腺树突状细胞的分化发育中发挥重要的调节作用。     

着丝粒蛋白B在基因组未复制的有丝分裂期细胞中的表达

着丝粒蛋白Ｂ（ＣＥＮＰ－Ｂ）主要位于染色体着丝粒区域的中心域，是一种ＤＮＡ结合蛋白，与染色体着丝粒、动粒的结构形成与功能实施有关。本研究结果显示咖啡因可以诱导ＣＨＬ细胞发生ＭＵＧ细胞。利用核酸杂交技术证明了在ＣＨＬ细胞中存在ＣＥＮＰ－Ｂ基因，并研究了咖啡因诱导ＭＵＧ细胞形成过程中ＣＥＮＰ－Ｂ的基因表达。发现ＣＥＮＰ－Ｂ在ＭＵＧ细胞整个过程中均有表达，表现出表达持续性。这与我们在Ｈｅｌａ细胞中发现的     

有丝分裂激酶Aurora-A活性调节的分子机制

Aurora-A是一种参与有丝分裂的丝氨酸/苏氨酸激酶,其过表达与肿瘤形成有关。Aurora-A激酶活性可以通过磷酸化、去磷酸化以及依赖蛋白酶体的降解等方式进行调节。TPX2(target protein for Xenopus kinesin-like protein 2)、1-2(Inhibitor 2)、Ajuba通过磷酸化Aurora-A将其活化,PP1(type-1 proteinphosphatase)等通过去磷酸化Aurora-A而抑制其活性,而且APC／C-Cdh1依赖的蛋白酶体等可以将其降解。了解和认识Aumra-A激酶活性调节的分子机制将有助于理解它在有丝分裂及肿瘤形成中的作用。     

有丝分裂纺锤体及中间小体提取方法的研究

目的 有丝分裂纺锤体和中间小体均是以在细胞有丝分裂进程中出现的微管为结构基础的暂时性结构,对细胞有丝分裂的顺利进行起着至关重要的作用.本研究旨在建立成熟高效的提取细胞内有丝分裂纺锤体和中间小体的方法.方法 通过细胞周期同步化方法使细胞内出现有丝分裂纺锤体或中间小体,运用低渗肿胀和甘油梯度离心的原理提取纺锤体和中间小体.结果 经Western Blot和细胞免疫荧光染色法鉴定,证实提取物中含有有丝分裂纺锤体和中间小体.结论 建立从人宫颈癌细胞Hela中提取有丝分裂纺锤体及中间小体的方法,可为鉴定有丝分裂纺锤体及中间小体上的蛋白分子垫定实验基础,同时也对研究肿瘤细胞有丝分裂的分子调控机制具有重要意义.     

人胎胸腺细胞的荧光染色观察

运用7690—Xu荧光染色法观察不同胎龄胎儿胸腺细胞的荧光映象,发现:不同胎龄人胎胸腺细胞悬液呈现相似形态和相似分布特征的8种异质性荧光图像的细胞.E花环实验和密度梯度离心分离结合7690—Xu荧光染色观察显示:墨黑核细胞和部分深蓝核细胞为分化早期细胞,淡桔黄核黄质细胞为分化晚期细胞,灰蓝核细胞和灰黄核细胞为处于分化中期的细胞.     

抗鼠T细胞受体抗体抑制胸腺细胞的迁出

目的 通过检测抗仓鼠T细胞受体抗体对胸腺T细胞输出的影响，进一步研究胸腺是提供外周免疫细胞输出的有关机理。方法 体内注射抗TCR抗体48h后FACS分析新迁出细胞在胸腺、淋巴结的表达。结果 小鼠成熟髓质区高表达T细胞受体的单阳性细胞数目成倍增加，同时皮质区低表达T细胞受体的不成熟双阳性细胞数目减少。成熟的单阳性胸腺细胞高表达归巢受体L-Selectin，表型分析(TCRαβ、CD69、HAS、Vβ7-integrin、Qa-2)显示增加的这群细胞为胸腺的新迁出细胞，此群成熟细胞的高表达，表明胸腺的细胞迁出受到了抑制。胸腺内注射异硫氰酸荧光素16h后，抗TCR抗体注射小鼠外周淋巴结及脾脏CD4^ 、CD8^ 新迁出细胞数量减少。结论 抗TCR抗体能抑制胸腺T细胞向外周迁移。     

胸腺细胞对胸腺基质细胞生长及功能的调节

目的研究胸腺细胞对不同亚群胸腺基质细胞生长及功能的调节作用。方法胸腺基质细胞增殖以３Ｈ－ＴｄＲ掺入法测定，ＩＬ－７活性以促ＩＬ－７依赖株法检测，其ｍＲＮＡ表达以ＲＴ－ＰＣＲ法检测。结果胸腺细胞与ＭＴＳＣ４细胞分别以８０∶１，４０∶１或２０∶１的比例共育时，能明显抑制ＭＴＳＣ４细胞的增殖，而培养上清中ＩＬ－７活性无明显改变；当两种细胞的比例为１０∶１或５∶１时，则对ＭＴＳＣ４细胞的增殖无任何影响，而培养上清中的ＩＬ－７活性明显升高。ＲＴ－ＰＣＲ证实，与胸腺细胞共育的ＭＴＥＣ１和ＭＴＳＣ４细胞，ＩＬ－７ｍＲＮＡ水平明显提高。结论胸腺细胞能促进ＭＴＥＣ１和ＭＴＳＣ４细胞分泌ＩＬ－７，对不同亚群基质细胞的生长和功能有不同调节作用     

Mitogenic effect contributes to increased virulence of Streptococcus suis sequence type 7 to cause streptococcal toxic shock-like syndrome

Streptococcus suis serotype 2 sequence type 7 strains emerged in 1996 and caused a streptococcal toxic shock-like syndrome in 1998 and 2005 in China. Evidence indicated that the virulence of S. suis sequence type 7 had increased, but the mechanism was unknown. The sequence type 7 strain SC84, isolated from a patient with streptococcal toxic shock-like syndrome during the Sichuan outbreak, and the sequence type 1 strain 31533, a typical highly pathogenic strain isolated from a diseased pig, were used in comparative studies. In this study we show the mechanisms underlying cytokine production differed between the two types of strains. The S. suis sequence type 7 strain SC84 possesses a stronger capacity to stimulate T cells, naive T cells and peripheral blood mononuclear cell proliferation than does S. suis sequence type 1 strain 31533. The T cell response to both strains was dependent upon the presence of antigen-presenting cells. Histo-incompatible antigen-presenting cells were sufficient to provide the accessory signals to naive T cell stimulated by the two strains, indicating that both sequence type 7 and 1 strains possess mitogens; however, the mitogenic effect was different. Therefore, we propose that the difference in the mitogenic effect of sequence type 7 strain SC84 compared with the sequence type 1 strain 31533 of S. suis may be associated with the clinical, epidemiological and microbiological difference, where the ST 7 strains have a larger mitogenic effect.     

Anti-seizure activity of the aqueous leaf extract of Solanum nigrum linn (solanaceae) in experimental animals

Background

Solanum nigrum is claimed in traditional medical practice, to be useful in the treatment of epilepsy in some parts of Nigeria.

Objectives

To study the anti-convulsant property of the aqueous extract of the leaves of S. nigrum in chicks, mice and rats.

Method

Aqueous extracts were administered intraperitoneally, at a pre-treatment time of 30 minutes, at graded doses and animals were challenged with different types of proconvulsants.

Results

The aqueous leaf extract produced a significantly (P<0.05) dose dependent protection against electrically-induced seizure in chicks and rats, pentylenetetrazole-induced seizure in mice and rats and picrotoxin-induced seizure in mice and rats. The anti-seizure property of the extract was potentiated by amphetamine.

Conclusion

The result obtained in this study suggests that the leaves of this plant may possess anti-convulsant property in chicks, mice and rats.     

Survivin-siRNA真核重组质粒对前列腺癌移植瘤的抑制作用

 目的：探讨survivin-siRNA真核重组质粒对前列腺癌移植瘤的抑瘤效应,并分析相关机制。方法：体外培养前列腺癌DU145细胞,将细胞注射到裸鼠背部皮下,待移植瘤直径达到8 mm时将瘤无菌取出,分成直径约2 mm的瘤块,手术植入另外的裸鼠皮下,将survivin-siRNA质粒或scrambled siRNA对照质粒电转染进行治疗,描记移植瘤生长曲线,并计算抑制率;通过HE染色、免疫组化染色及TUNEL染色观察survivin-siRNA对移植瘤影响。结果：成功构建裸鼠前列腺癌移植瘤模型。与mock和scrambled siRNA组相比,survivin-siRNA抑瘤率分别为两对照组的61.81%和62.87%;免疫组织化学染色发现：survivin-siRNA质粒明显抑制细胞内survivin表达;TUNEL染色证实治疗组肿瘤细胞凋亡明显增加。结论：重组质粒survivin-siRNA明显抑制DU145细胞移植瘤的生长,抑制内源性 survivin 蛋白的表达并促进细胞凋亡,针对survivin靶点的基因治疗前景广阔。     

粉防己碱与表阿霉素联用对表阿霉素在荷瘤裸鼠血、心与肿瘤组织浓度的影响

目的:研究多药耐药(MDR)裸鼠移植瘤模型中,粉防己碱以不同频率和时间间隔给药对抗癌药物表阿霉素进入肿瘤组织细胞能力的影响。方法:裸鼠背部皮下接种人口腔上皮癌(耐药株KBV和非耐药株KB)细胞,按粉防己碱给药次数和提前表阿霉素给药的时间,试验分组为:组1(单次,提前48h)、组2(单次,提前24h)、组3(单次,提前2h)、组4(两次,提前72h和24h)、组5(两次,提前48h和24h)、组6(两次,提前24h和2h)、耐药株对照、敏感株对照。粉防己碱(50mg.kg-1)腹腔注射给药,表阿霉素(8mg.kg-1)尾静脉注射给药。分别于表阿霉素给药后3小时取血、解剖取心脏和肿瘤组织,HPLC法测定血液和心脏组织中表阿霉素的含量,质谱法测定肿瘤组织样品的表阿霉素的含量。结果:耐药组裸鼠表阿霉素血液含量为7.2±1.1 ng.ml-1,心脏组织中为60.1±20.2ng.g-1,与敏感组的测定值一致,但肿瘤中的含量只有8.4±3.9ng.g-1,与耐药组相比有统计学差异(P<0.05),组1、2、3、5肿瘤组织中表阿霉素含量同耐药对照组相比无统计学意义,相互之间也无显著差异。组4(-48,-24h)的肿瘤组织浓度高于耐药对照组,同时心脏组织中的含量比耐药对照组的低,有统计学意义。结论:粉防己碱提前48h或24h给药似有较好的抗MDR效果,且既能提高耐药肿瘤中表阿霉素的浓度,又减少表阿霉素对心肌的药源性损伤,具有潜在的临床意义。     

骨髓基质细胞HS-分泌物白细胞介素-对人急性髓细胞性白血病细胞系HL-0抗凋亡的作用

目的 探讨骨髓基质细胞HS-分泌物白细胞介素-6(IL-6)急性髓细胞性白血病（AML）细胞系HL-0增殖与凋亡的影响。方法 外培养HL-60细胞与HS-5细胞,构建共培养体系,应用扫描电子显微镜、ELISA法、细胞计数试剂盒8（CCK-8）法、AnnexinV-FITC/PI 双染流式细胞术、Real-time PCR和Western blotting技术分别检测HL-60细胞增殖和凋亡的变化;将不同组的HL-60细胞分别接种于BALB/c裸鼠,观察并记录成瘤情况。结果 骨髓基质细胞HS-5分泌的IL-6能够促进HL-60细胞增殖、抑制其凋亡,并下调HL-60细胞中的促凋亡基因Bax的表达,上调抗凋亡基因Bcl-2的表达。共培养组HL-60细胞在BALB/c裸鼠中成瘤能力最强,HL-60细胞单独培养组成瘤能力最弱。结论 骨髓基质细胞HS-5促进HL-60细胞增殖、抑制其凋亡的部分作用机制是通过分泌IL-6实现的。     

Studies on the T cell dependence of natural IgM and IgG antibody repertoires in adult mice

The present experiments address the thymic dependence of IgM and IgG natural antibody repertoires in adult euthymic and athymic BALB/c mice, as well as in athymic animals reconstituted with a fixed number of syngeneic T cells. Within 3 weeks of the transfer of 10⁷ syngeneic splenic T lymphocytes to athymic mice, the T cell compartment is essentially reconstituted in the peritoneal cavity (up to 80% of the numbers in euthymic animals), but is only 10–20% of controls in the spleen and lymph nodes. Early after transfer, there is an increase in the numbers of activated B cells and of immunoglobulin-secreting cells in the spleen, and within 1–2 weeks, the serum concentrations of IgG₁ and IgG_2a are fully reconstituted to control levels (30–40-fold increased). Multiparametric analyses of serum IgM and IgG repertoires revealed that euthymic and athymic mice share essentially all natural antibody reactivities towards syngeneic extracts of liver and muscle. When tested at the same immunoglobulin concentrations, however, nude sera consistently show higher values of reactivity in all detectable bands. The transfer of 10⁷ splenic T cells into athymic mice results in a general decrease of serum IgM reactivities, some of which become undetectable, and in alterations of the serum IgG repertoire as early as 1 week, and for at least 4 weeks after transfer. T cell transfer, however, fails to restore the euthymic IgM and IgG repertoires within 4 weeks. The present observations demonstrate that, after limited T cell reconstitution of nude mice, there is a rapid and quantitatively important increase of serum IgG₁ and IgG_2a production; the serum IgM reactivity repertoire is qualitatively similar in euthymic and athymic animals, but is generally decreased by T cell activity; and the serum IgG repertoire, which is qualitatively similar in euthymic and athymic animals, is amplified by T cell activity and partially altered by T cell transfer into athymic animals. These results raise questions on the mechanisms of B cell activation and natural antibody repertoire selection in T cell-deficient adult individuals.     

李斯特菌感染对小鼠APC活性的影响

目的：通过对转基因鼠脾T细胞的分化研究,探讨感染初期APC的活性。方法：细胞因子ELISA测定转基因鼠脾T细胞产生的IFN-γ及IL-4,FACS鉴定T细胞为TCR-TG的T细胞（Vβ8.2品系）。结果：感染鼠的APC诱导TG鼠的T细胞向Th1方向分化,未感染鼠的APC诱导TG鼠的T细胞向Th2方向分化,IL-12和IL-4可影响APC对T细胞分化的诱导。结论：感染和外来细胞因子影响APC的提呈诱导活性。     

Absolute dependence of T cell receptorhi cell generation and relative dependence of T cell receptorint cell generation on the thymus

Recent evidence indicates that conventional T cells are generated by the mainstream of T cell differentiation in the thymus and acquire a high density of T cell receptor expression (i.e. TCR^hi). In contrast, primordial T cells (or NK1.1⁺ T cells) are generated by the extrathymic pathways or an alternative intrathymic pathway and express an intermediate density of TCR (i.e. TCR^int). To obtain further evidence, it was examined how thymus grafting influenced the distribution of T cell populations in athymic nude mice. When BALB/c nu/nu mice were engrafted with thymocyte-depleted BALB/c+/+ fetal thymi, two changes emerged after grafting: nude mice generated TCR^hi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin-2 receptor β chain⁺ TCR^int cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin α induced a slight expansion of CD3^int cells in nude mice. To examine a possible interaction of TCR^int cells with TCR^hi cells in the periphery, B6 nu/nu mice (Ly5.2⁺) were injected with TCR^hi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCR^int (Ly5.2⁺) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCR^hi cells is absolutely dependent on the thymus and that TCR^int cells expand under the influence of the thymus (humoral) and due to interaction with thymus-derived conventional T cells.     

硒对正常人细胞免疫功能的影响

本实验在体外观察了硒对正常人淋巴细胞转化率、淋巴细胞产生IL-2能力以及淋巴细胞对IL-2反应性的影响。结果表明硒能增强人淋巴细胞转化率、淋巴细胞产生IL-2能力,但不能提高淋巴细胞对IL-2反应性。提示硒可通过增强淋巴细胞产生IL-2能力而增强人体免疫系统抗肿瘤能力。