c-myc反义寡核苷酸对缺氧内皮细胞条件培养液刺激的肺血管平滑肌细胞增殖的影响

目的探讨ｃ－ｍｙｃ反义寡核苷酸（ＯＤＮｓ）对缺氧内皮细胞条件培养液（ＨＥＣＣＭ）刺激的肺血管平滑肌细胞（ＳＭＣ）增殖的影响。方法制备ＨＥＣＣＭ，用其刺激肺动脉ＳＭＣ后，Ｎｏｒｔｈｅｒｎ杂交分析ｃ－ｍｙｃ基因表达，３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入试验及细胞生长计数分析ＳＭＣ增殖情况。结果ＨＥＣＣＭ显著刺激ＳＭＣ增殖及ｃ－ｍｙｃ基因表达增强，反义ＯＤＮｓ显著下调ＨＥＣＣＭ刺激的ｃ－ｍｙｃ表达，显著抑制ＨＥＣＣＭ刺激的ＳＭＣ增殖。同义ＯＤＮｓ无上述作用。结论ＨＥＣＣＭ可能通过刺激ＳＭＣ之ｃ－ｍｙｃ表达而使ＳＭＣ增殖，反义ＯＤＮｓ通过下调ｃ－ｍｙｃｍＲＮＡ表达而抑制ＨＥＣ－ＣＭ刺激的ＳＭＣ增殖。     

胎肝提取物对BEL—7402肝癌细胞的影响

从４个月胎肝中制备提取物，采用３Ｈ－ＴｄＲ掺入，ＭＴＴ比色法和细胞形态学等技术观察了胎肝提取物对ＢＥＬ－７４０２肝癌细胞的影响。１％和２％ＤＭＳＯ对细胞ＤＮＡ合成的抑制率为５６％和５６％；对线粒体脱氢酶的抑制率为４３％和４８％；形态学改变主要表现为有丝分裂相减少，胞质出粗细不等的突起，相互连成网状结构。     

人胎肝刺激物质的纯化与活性测定

目的本研究对人胎肝刺激物质的纯化进行了研究，并对其活性测定方法进行了探讨．方法ｈＨＳＳ经离子交换层析，非变性聚丙烯酰胺凝胶电泳和ＩＳＣＯＣｏｎｃｅｎｔｒａｔｏｒ进行了纯化，经ＳＤＳ聚丙烯酰胺凝胶电泳进行了分子量测定，ｈＨＳＳ的活性由３ＨＴｄＲ掺入法（改进）和ＥＬＩＳＡ法进行了测定．结果ｈＨＳＳ的纯化程度随着纯化步骤的进行而逐渐增加．经过许多步骤后，非变性ＰＡＧＥ的主带显示了较高的活性和纯化程度，并且非变性ＰＡＧＥ的一条主带在ＳＤＳＰＡＧＥ中显示两条带．结论ｈＨＳＳ的分子量约为７０ＫＤ左右，它由１８ＫＤ和５３ＫＤ两种亚基组成．ｈＨＳＳ的活性由３ＨＴｄＲ掺入和ＥＬＩＳＡ两种方法测定，并且两种方法具有较好的一致性．     

不饱和脂肪酸对L-02和HLF细胞增殖及合成细胞外基质的影响

目的探讨不饱和脂肪酸对肝细胞和成纤维细胞增殖及合成细胞外基质的影响．方法以ＭＴＴ法、透明质酸（ＨＡ）ＲＩＡ法及３Ｈ脯氨酸掺入法，观察４种不同浓度的油酸、亚油酸和花生四烯酸对无血清培养正常成人肝细胞（Ｌ０２）、人胚肺成纤维细胞（ＨＬＦ）增殖及合成细胞外基质（ＥＣＭ）的影响．结果油酸、亚油酸可影响ＨＬＦ细胞和（或）Ｌ０２细胞增殖，并促进其合成胶原和ＨＡ，而花生四烯酸对其增殖及合成ＥＣＭ的影响不明显．结论肝内游离的不饱和脂肪酸增多可促进肝纤维化的发生和发展．     

改良成人胃粘膜上皮细胞原代培养

建立成人胃粘膜上皮细胞原代培养方法。方法：采用胰酶和胶原酶冷消化法消化分离手术切除的成人胃粘膜，细胞培养于含２０％小牛血清的ＤＭＥＭ／Ｆ１２培养液中。结果：细胞于种植２４ｈ后开始生长，３天长成片状，相差显微镜下可见９０％以上的细胞具有上皮细胞特征。免疫组化显示９０％以上的细胞上皮角蛋白染色、上皮膜抗原阳性。ＭＴＴ比色法显示活细胞数于第４天达到高峰。＾３Ｈ－胸腺嘧啶核苷（ＴｄＲ）掺入法显示细胞具有合     

肝干细胞的分离培养

分离鼠胎肝细胞 ,观察人肝细胞生长肽 (ＨＧＰ)和小鼠白血病抑制因子 (ｍＬＩＦ)对鼠胎肝细胞生长和肝干细胞集落形成的影响。从大鼠胚胎 ( 12日龄 )肝中分离肝细胞 ,采用ＭＴＴ比色法、3Ｈ ＴｄＲ掺入法和碱性磷酸酶 (ＡＫＰ)检测法。结果表明 ,人ＨＧＰ能剂量依赖性地促进胎肝细胞的ＤＮＡ合成和增殖 ,50 0～ 2 0 0 0Ｕ/ｍｌ的ｍＬＩＦ能明显促进肝干细胞集落的生长和维持未分化状态。肝干细胞培养基的组成为 :5%ＦＢＳ(国产或进口 ) ,10 %国产ＮＣＳ ,0 .14ｍＭ的β ＭＥ ,4 0ｎｇ/ｍｌ伴白蛋白 ,10ｎｇ/ｍｌ的人ｂＦＧＦ ,0 .5～ 1.0 μ…     

去甲肾上腺素,人参皂甙和硝苯啶对大鼠动脉平滑肌细胞增殖和D…

目的 血管的老化与血管平滑肌细胞（ＶＳＭＣ）的异常增殖有关，我们拟探讨其增殖机制并寻求有效防治措施。 方法 应用噻唑蓝（ＭＴＴ）染色法和＾３Ｈ－ＴｄＲ掺入法观察去甲肾上腺素（ＮＥ）、硝苯啶（Ｎｉｆ）以及人参皂甙（Ｇｉｎ）对老年（１８月龄）和青年（３月龄）大鼠培养的主动脉平滑肌细胞（ＡＳＭＣ）增殖和细胞内ＤＮＡ合成的影响。 结果 （０．１￣５）×１０＾－５ｍｏｌ／Ｌ的ＮＥ可明显促进ＡＳＭＣ呈剂量依赖     

表皮生长因子对人衰老成纤维细胞EGFR基因及HER－2基因表达的诱导

人胚肺成纤维细胞（２ＢＳ）的３Ｈ脱氧胸苷（ＴｄＲ）掺入率，衰老细胞明显低于中年和年轻细胞（Ｐ＜０．０１）。表皮生长因子（ＥＧＦ）对三种细胞的３Ｈ－ＴｄＲ掺入均有促进作用，衰老细胞的反应性明显降低。Ｎｏｒｔｈｅｒｎ及斑点杂交法显示衰老细胞的ＨＥＲ－２ｍＲＮＡ较年轻细胞明显降低，ＥＧＦ受体ｍＲＮＡ水平仅在极端衰老时才下降，ＥＧＦ可诱导年轻细胞ＨＥＲ－２基因表达，而衰老细胞却难以诱导。     

贮脂细胞和肝细胞胶原合成能力的比较

目的为明确肝纤维化过程中细胞外基质（ｅｘｔｒａｃｅｌｕｌａｒｍａｔｒｉｘ，ＥＣＭ）的细胞来源以及贮脂细胞（ｆａｔｓｔｏｒｉｎｇｃｅｌ，ＦＳＣ）和肝细胞（ｈｅｐａｔｏｃｙｔｅ，ＨＣ）在肝纤维化胶原合成中的作用．方法用胶原酶和链霉蛋白酶Ｅ消化大鼠肝脏，结合密度梯度离心法分离培养大鼠ＨＣ和ＦＳＣ，在此基础上，用３Ｈ脯氨酸掺入结合胶原酶消化法和斑点杂交法，观察ＨＣ和ＦＳＣ胶原的合成及Ⅰ，Ⅲ，Ⅳ型前胶原基因的表达．结果发现体外培养的ＦＳＣ合成胶原的能力较ＨＣ强，约为ＨＣ的两倍（Ｐ＜００５）；ＦＳＣ胶原基因表达的水平同样较ＨＣ高（Ｐ＜００５）．结论ＦＳＣ在ＥＣＭ胶原合成中起主要作用，但ＨＣ的作用也不容忽视．     

低氧对肺血管周细胞增殖及分化的影响

目的研究低氧对肺血管周细胞增殖及分化的影响,探讨低氧性肺动脉高压时无肌型肺动脉肌化的细胞来源。方法实验分为４组：直接低氧组（Ｈ组）,常氧组（Ｎ组）,低氧内皮细胞条件培养液组（ＨＥＣＣＭ组）,常氧内皮细胞条件培养液组（ＮＥＣＣＭ组）。应用细胞培养、３Ｈ胸腺嘧啶核苷（３ＨＴｄＲ）、免疫细胞化学、图像分析等技术,研究低氧对肺血管周细胞３ＨＴｄＲ的掺入量、增殖细胞核抗原（ＰＣＮＡ）、α平滑肌肌动蛋白（αＳＭＡｃｔｉｎ）表达的影响。结果Ｈ组周细胞３ＨＴｄＲ的掺入量、ＰＣＮＡ及αＳＭＡｃｔｉｎ的表达量分别是Ｎ组的２．１６倍（Ｐ＜０．０１）、１．１６倍（Ｐ＜０．０１）及１．１１倍（Ｐ＜０．０５）,ＨＥＣＣＭ组周细胞３ＨＴｄＲ的掺入量、ＰＣＮＡ及αＳＭＡｃｔｉｎ的表达量分别是ＮＥＣＣＭ组的１．８倍（Ｐ＜０．０１）、１．１５倍（Ｐ＜０．０１）及１．１２倍（Ｐ＜０．０５）。结论低氧可直接或刺激内皮细胞分泌某些细胞因子促进肺血管周细胞增殖、并向平滑肌样细胞分化。肺血管周细胞的增殖、向平滑肌样细胞分化是低氧性肺动脉高压无肌细动脉肌化的重要细胞来源     

Mixed lymphocyte reactions evaluated by means of bromodeoxyuridine incorporation

The mixed lymphocyte reactions are usually performed by the uptake of 3H thymidine (3H TdR) in the study of histocompatibility for allogeneic bone marrow transplantation. Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be used as an alternative marker of proliferation. In this study we compared the evaluation of the proliferative activity of alloreactive lymphocytes in MLR by 3H TdR and BrdUrd incorporation in flow cytometry. The results show that BrdUrd is able to recognize proliferative activity at least 24-48 hours before 3H TdR, and allows discrimination of proliferation of a few cells despite its proximity to the autologous controls. However, the large quantity of cells needed for every combination is presently a disadvantage of the method.     

The relationship between humoral stimulating activity and colony stimulating factor

Humoral stimulatory activity (HSA) is detected in the sera of neutropenic rodents and humans by its effects on in vitro marrow cell [3H] TdR incorporation. Increased levels of CSA are also found in such sera; however, the relationship between these two activities has not been established. To examine this question, sera were obtained from mice, rats and humans at the peak occurrence of HSA following cyclophosphamide administration and were incubated with rabbit antiserum directed against mouse L-cell CSF. Anti-CSF neutralized the HSA effect on species-specific marrow cell [3H] TdR incorporation relative to the effects of normal sera with an efficiency of anti-CSF produced graded neutralization of the human HSA effect on human marrow cell proliferation. This neutralizing effect of anti-CSF on human HSA was obrogated by prior incubation of anti-CSF with sheep-anti-rabbit-IgG (S-anti-R-IgG) serum. The human serum activities on marrow cell proliferation, as determined by the [3H] TdR incorporation assay, correlated closely with their effects on human granulocyte colony formation in agar culture. These data indicate that human and rodent drug-induced HSA are cross-reactive with CSF as measured by a simple proliferation assay.     

含弓形虫ROP1基因真核表达重组质粒DNA免疫小鼠的研究Ⅴ.IFN-γ基因真核表达重组质粒的构建及其在DNA免疫中基因佐剂的作用

目的　构建ＩＦＮ－γ基因真核表达重组质粒作为基因佐剂,观察其与ｐｃＤＮＡ３－ＲＯＰ１（ｐｃ－ＲＯＰ１） ＤＮＡ共同免疫小鼠所诱导的免疫应答。方法　将ＩＦＮ－γ基因片段定向插入真核表达载体ｐｃＤＮＡ３,双酶切鉴定,获得ｐｃＩＦＮ 重组子;碱裂解法大量制备,经肌肉注射免疫ＢＡＬＢ／ｃ小鼠,每只鼠注射ｐｃＩＦＮ、ｐｃ－ＲＯＰ１ 各１００μｇ,两周后同量加强免疫一次,以ｐｃＤＮＡ３空质粒及生理盐水组为对照。分别于免疫后第３０ 天、５０ 天、７０天共三次用ＭＴＴ法测定小鼠脾脏Ｔ淋巴细胞增殖活性及ＮＫ细胞活性;双夹心ＥＬＩＳＡ 测定血清细胞因子ＩＦＮ－γ、ＩＬ－２及酶法测定ＮＯ含量;ＥＬＩＳＡ法测定ＩｇＧ抗体滴度。结果　构建成功的作用下,该两项指标均明显提高,且ＩＦＮ－γ、ＩＬ－２ 及ＮＯ水平均较不加佐剂组显著提高（Ｐ＜ ０．０１）;而对ＩｇＧ抗体滴度无显著影响（Ｐ＞ ０．０５。结论　ＩＦＮ－γ基因佐剂具有协同ｐｃ－ＲＯＰ１ＤＮＡ免疫的作用,可增强免疫鼠细胞免疫应答,ＩＦＮ－γ、ＩＬ－２细胞因子及ＮＯ的产生     

Isolation and functional identification of a novel human hepatic growth factor: hepatopoietin Cn

Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [(3)H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. CONCLUSION: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration.     

恶性疟原虫FCC-1/HN株pcDNA_3-Pfs25重组质粒在小鼠体内的免疫应答

目的　探讨恶性疟原虫ＤＮＡ 疫苗在小鼠体内的免疫反应。方法　将恶性疟原虫ＦＣＣ－ １／ＨＮ株有性期阶段的重组质粒ｐｃＤＮＡ３－ Ｐｆｓ２５ 经骨骼肌途径注射ＢＡＬＢ／ｃ小鼠,对注射部位的骨骼肌进行了预处理,即于注射前７ｄ 先在左后肢股四头肌注射０．５％ 盐酸布比卡因５０μｌ,注射深度为２ｍ ｍ 。观察免疫后不同时间点小鼠血清ＩｇＧ抗体滴度、脾淋巴细胞增殖反应、ＣＤ４＋ ／ＣＤ８＋ Ｔ细胞亚群比值和ＮＫ 细胞杀伤活性的变化。结果　１）重组质粒ｐｃＤＮＡ３－ Ｐｆｓ２５ 经肌肉注射途径免疫小鼠后,机体ＩｇＧ抗体滴度增高、特异性Ｔ淋巴细胞增殖反应增强、ＣＤ８＋ Ｔ细胞亚群比值增高以及ＮＫ细胞杀伤活性增强。２）肌肉注射为一有效的ＤＮＡ疫苗免疫途径。结论　采用编码有性期基因的重组质粒ｐｃＤＮＡ３－ Ｐｆｓ２５ 经骨骼肌注射途径免疫小鼠,能诱导机体有效的体液免疫、细胞免疫和ＮＫ细胞杀伤活性。本研究为恶性疟ＤＮＡ疫苗的研究提供了免疫学实验依据     

苦参合剂对隐孢子虫病儿童免疫功能的影响

目的　观察苦参合剂对隐孢子虫病儿童免疫功能的影响。方法　用ABC法和3H-TdR掺入法检测T淋巴细胞亚群及淋巴细胞转化率,用单向琼脂扩散法检测免疫球蛋白,对隐孢子虫病儿童苦参合剂治疗前后的免疫功能进行测定。结果　患儿Tcell、Th值降低,淋巴细胞转化受抑,IgG升高。苦参合剂治疗后,临床腹泻症状消失,排虫转阴。Tcell、Th淋巴细胞转化值显著升高。结论　苦参合剂对隐孢子虫的感染有较好的驱虫与增强患儿细胞免疫力的作用。     

Endocrine and paracrine control of Leydig cell steroidogenesis and proliferation in the wall lizard: an in vitro study

The present in vitro study, for the first time, demonstrates the endocrine as well as paracrine control of Leydig cell steroidogenesis and proliferation in the wall lizard Hemidactylus flaviviridis. Unlike mammals, Leydig cell activity in the wall lizard seems to be directly controlled by ovine follicle-stimulating hormone (FSH)-like molecule, since FSH increased the testosterone production and tritiated thymidine ([(3)H]TdR) incorporation by Leydig cells. In addition, Sertoli cell paracrine factor or factors play important roles in controlling Leydig cell activities as non-activated Sertoli cell-conditioned medium (SCCM) alone stimulated testosterone production by both non-activated and FSH-preactivated Leydig cells. As far as the proliferation was concerned, non-activated SCCM did not influence [(3)H]TdR uptake by non-activated or FSH-preactivated Leydig cells, while FSH-preactivated SCCM was able to stimulate proliferation of activated Leydig cells. It may be concluded that FSH, besides directly controlling, also regulates Leydig cell activities indirectly through stimulating the secretion of Sertoli cell paracrine factors. Moreover, steroidogenic factor is different from mitogenic factor because non-activated Leydig cells were responsive to steroidogenic factor but nonresponsive to mitogenic factor.     

Balb/C小鼠免疫系统结构与功能的增龄性变化

目的 观察不同月龄Ｂａｌｂ／Ｃ小鼠免疫系统结构珉 改变,以增进对衰老免疫学机制报了解。方法 采用＾３Ｈ胸腺嘧核苷（＾３Ｈ－ＴｄＲ）掺入法,噻唑蓝（ＭＴＴ法）,白细胞介素２（ＩＬ－２）依赖细胞株（ＣＴＬＬ－２）测定等实验,检测１、５、１０和１９月龄小鼠脾细胞表面抗原表达及免疫功能,并用苏木精伊红（ＨＥ染色检测胸腺和脾脏的组织学变化。结果 随着增龄,小鼠胸腺皮、髓质比例砬小,界限消失,有分泌液泡形成,     

RRR-α-tocopheryl succinate inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

AIM: To investigate the effects of growth inhibition ofhuman gastric cancer SGC-7901 cell with RRR-α-tocopherylsuccinate (VES), a derivative of natural Vitamin E, viainducing apoptosis and DNA synthesis arrest.METHODS: Human gastric cancer SGC-7901 cells wereregularly incubated in the presence of VES at 5, 10 and20mg@ L 1(VES was dissolved in absolute ethanol anddiluted in RPMI 1640 complete condition mediacorrespondingly to a final concentration of VES and 1mL@L-1 ethanol), succinic acid and ethanol equivalents asvehicle (VEH) control andcondition media only asuntreated (UT) control. Trypan blue dye exclusionanalysis and MTT assay were applied to detect the cellproliferation. 37kBq of tritiated thymidine was added tocells and [3H] TdR uptake was measured to observe DNAsynthesis. Apoptotic morphology was observed byelectron microscopy and DAPI staining. Flow cytometryand terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) assay were performed to detectVES-triggered apoptosis.RESULTS: VES inhibited SGC-7901 cell growth in a dose-dependent manner. The growth curve showed suppressionby 24.7%, 49.2% and 68.7% following 24h of VEStreatment at 5, 10 and 20 mg@L 1, respectively, similar tothe findings from MTT assay. DNA synthesis wasevidently reduced by 35%, 45% and 98% after 24h VEStreatment at 20 mg@ L-1 and 48h at 10 and 20 mg@ L 1,respectively. VES induced SGC-7901 cells to undergoapoptosis with typically apoptotic characteristics,including morphological changes of chromatincondensation, chromatin crescent formation/margination,nucleus fragmentation and apoptotic body formation,typical apoptotic sub-G1 peak by flow cytometry andincrease of apoptotic cells by TUNEL assay in which 90%of cells underwent apoptosis after 48h of VES treatment at20 mcg@L-1.CONCLUSION: VES can inhibit human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesisarrest. Inhibition of SGC-7901 cell growth by VES is dose-and time-dependent. Therefore VES can function as apotent chemotherapeutic agent against human gastriccarcinogenesis.     

SPECIFIC INHIBITION OF TUMOR CELL DNA SYNTHESIS In Vitro BY LYMPHOCYTES FROM PERITONEAL EXUDATE OF IMMUNIZED SYNGENEIC GUINEA PIGS

Tumor immunity to a transplantable diethylnitrosamine-induced hepatoma in inbred guinea pigs has been found to be immunologically specific and cell mediated. We have investigated this cellular immunity using a quantitative, reproducible, and simple assay based on the ability of leucocytes to inhibit the incorporation of tritiated thymidine (TdR(3)H) by tumor cells. Tumor cell suspensions were obtained from the ascites form or tissue culture monolayers of the hepatoma. Cells from the peritoneal exudate of immunized guinea pigs inhibited tritiated thymidine uptake by tumor target cells to a significantly greater degree than cells from the peritoneal exudate of unimmunized animals. Immune lymph node, peripheral blood, and spleen leucocytes were not inhibitory. The assay was sufficiently sensitive to detect relatively weak tumor immunity. The in vitro inhibition was correlated directly with the presence of delayed hypersensitivity and/or inhibition of tumor cell growth in local passive transfer studies. Irradiation of peritoneal exudate cells (3000 R) blocked their inhibitory effects on tumor cells. Fractionation of the peritoneal exudate cells by centrifugation in zones of bovine serum albumin of different density also revealed the lymphocytes to be responsible for the specific inhibitory effects whereas macrophages inhibited in an immunologically nonspecific fashion.