噻唑蓝技术测定供移植用大鼠Leydig氏细胞的活力

目的：探讨大鼠Ｌｅｙｄｉｇ氏细胞的活力。方法：用噻唑蓝（ＭＴＴ）技术和台盼兰染色两种方法，对移植前大鼠Ｌｅｙｄｉｇ氏细胞作活力测定。结果：噻唑蓝比色法是一种敏感、可靠、简单的细胞活力测定技术。结论：细胞ＭＴＴ测定值高新体外攻体内的存活能力也强。     

雌雄激素对前列腺基质细胞增殖的影响

目的 探讨雌雄激素对良性前列腺增生发病机理中的作用。方法 体外分离和培养人前列腺基质细胞，应用ＭＴＴ比色法观察雌雄激素和雄激素以及雌激素和雄激素相互作用对基质细胞增殖的作用。结果 第三代基质细胞的纯化率达到９５％以上。部分纤维母细胞在培养过程中转变为平滑肌细胞。雌激素使前列腺基质细胞的增殖超过对照组１１４％～１２２％，雄激素使用前腺基质细胞的地殖超过对照组１１４％～１３３％（Ｐ〈０．０１），与单一     

外源性胃泌素在胃癌细胞增殖和凋亡中的作用

我们应用ＭＫＮ45胃癌细胞株通过四唑蓝 (ＭＴＴ)比色分析法及流式细胞仪观察胃泌素在细胞增殖、凋亡中的作用 ,同时观察丙谷胺对胃泌素的拮抗作用 ,并与 5 氟尿嘧啶 (5 Ｆｕ)作比较 ,结果报道如下。一、材料与方法1.细胞培养 :ＭＫＮ45胃癌细胞株(上海市消化疾病研究所提供 )培养于ＲＰＭＩ 164 0培养液中 ,加入体积分数为10 %小牛血清 ,置 3 7℃ 5 %ＣＯ2 恒温培养箱 ,隔天换液 ,3ｄ传代。2 .试剂配制及实验分组 :用 5 %的小牛血清的 164 0液将胃泌素 (上海丽珠东风生物技术公司 )配为 2 5ｍｇ/Ｌ浓度 ,丙谷胺 (江苏金坛制药厂 )浓度为 …     

肿瘤坏死因子α和干扰素α对膀胱癌患者LAK细胞增殖和细…

为探讨联合应用白细胞介素２（ＩＬ－２）和干扰素（ＩＦＮ）或肿瘤坏死因子（ＴＮＦ）对膀胱癌患者ＬＡＫ细胞的增殖和细胞毒作用的影响，采用淋巴细胞分离液梯度离心法从２１例膀胱癌患者外周血分离ＬＡＫ细胞并用ＩＬ－２激活培养，细胞于９６孔细胞培养板用细胞计数方法观察了不同浓度ＴＮＦα和ＩＦＮα对细胞增殖的影响，用膀胱癌细胞系ＢＩＵ－８７和ＥＪ细胞作为靶细胞，用ＭＴＴ法测定ＬＡＫ细胞的细胞毒。结果：ＴＮＦα呈     

小鼠胚胎睾丸Leydig细胞培养、纯化及其功能研究

目的:探讨小鼠胚胎睾丸Leyd ig细胞体外培养、鉴定、纯化的方法,并进行形态学观察、分泌睾酮能力等生物学特性检测。方法:选择胎龄16 d的胎鼠睾丸,0.03%胶原酶Ⅰ消化,3β-羟基类固醇脱氢酶(HSD)染色鉴定及纯度测定,锥虫蓝染色检测细胞活率。放免法测定Leyd ig细胞不同培养时间及密度下分泌睾酮的水平。结果:Leyd ig细胞培养前和培养72 h后纯度分别为(45.10±1.66)%和(81.17±2.32)%;培养液中可检测到睾酮,睾酮水平与Leyd ig细胞数、培养时间相关,单个Leyd ig细胞睾酮分泌能力逐日下降。结论:该法分离的胚胎Leyd ig细胞纯度较高,生长性好,保持增殖和分泌睾酮的生物学特性,可应用于相关的研究。     

细胞素及合用异博定对人乳腺癌细胞多药耐药性逆转作用的研究

我们利用人乳腺癌耐药细胞系以流式细胞术及噻唑蓝 (ＭＴＴ)法检测肿瘤坏死因子 (ＴＮＦ)、白细胞介素 (ＩＬ) 2、α 干扰素 (α ＩＦＮ)单用及与异博定合用对细胞耐药性的调节作用 ,结果报道如下。一、材料与方法1.细胞 :人乳腺癌亲代ＭＣＦ 7细胞及对阿霉素耐药的ＭＣＦ 7/ＡＤＲ细胞来源于美国国立癌症研究院 ,常规培养。2 .ＭＣＦ 7/ＡＤＲ细胞的抑制率测定 :将细胞调整至 3× 10 8个 /Ｌ浓度 ,取2 0 0 μｌ加入 96孔板中过夜 ,去掉培养基 ,加入含不同药物浓度的新培养基 ,培养 48ｈ收集细胞 ,ＭＴＴ法检测半抑制浓度 (ＩＣ50 )。3.细…     

表皮生长因子对子宫内膜细胞体外增殖的影响

本研究观察了表皮生长因子（ＥＧＦ）在体外对子宫内膜上皮细胞及基质细胞增殖的影响。采用酶消化加物理方法分离人子宫内膜上皮细胞及基质细胞,分别在体外培养,细胞汇合后消化,一部分用盖片法培养,用特异性抗体鉴定细胞纯度,另一部分做细胞增殖实验。在细胞中加入不同浓度的ＥＧＦ,培养２４、４８、７２小时,用ＭＴＴ法及流式细胞仪测定ＥＧＦ对子宫内膜上皮细胞及基质细胞增殖的作用。结果：ＥＧＦ浓度为５．０及１０．０ｎｇ／ｍｌ时,明显刺激子宫内膜上皮细胞及基质细胞的增殖,ＭＴＴ与流式细胞仪两法一致。ＥＧＦ不刺激细胞凋亡。结论：采用酶消化结合物理方法分离的人子宫内膜基质细胞及上皮细胞,方法简便、快速,细胞纯度高,在体外生长良好,可用于体外研究着床及异位子宫内膜生长的机制。当ＥＧＦ浓度为５．０及１０．０ｎｇ／ｍｌ时,确实刺激子宫内膜细胞的增殖。     

减少内源性脂肪酸合成对乳腺癌细胞生长的影响

内源性脂肪酸由位于细胞胞浆中的脂肪酸合成酶 (ｆａｔｔｙａｃｉｄｓｙｎｔｈａｓｅ,ＦＡＳ)催化合成 ,Ｃｅｒｕｌｅｎｉｎ是ＦＡＳ的强抑制剂。我们应用四甲基唑蓝 (ＭＴＴ)方法在体外研究Ｃｅｒｕｌｅｎｉｎ对乳腺癌细胞生长的抑制作用及ＦＡＳ直接产物软脂酸对Ｃｅｒｕｌｅｎｉｎ抑癌作用的影响 ,现报道如下。一、材料与方法1.四甲基唑蓝 :华美生物工程公司 ;Ｃｅｒｕｌｅｎｉｎ :美国Ｓｉｇｍａ公司产品 ,用二甲基亚砜配成 5 ｇ/Ｌ的使用溶液。2 .细胞培养 :人乳腺癌细胞株ＭＣＦ 7由中国科学院上海细胞研究所提供 ,常规单层贴壁传代培…     

天然与合成的甾本激素对培养的人子宫动脉平滑肌细胞增殖的影响

应用＾Ｈ３－ＴｄＲ掺入和四甲基偶氮唑盐（ＭＴＴ）法观察了天然与合成甾体激素对培养的人子宫动脉平滑肌细胞（ＨＵＡＳＭＣ）增殖的影响，结果：雌激素对ＨＵＡＳＭＣ增殖无明显影响，孕激素可促进ＨＵＡＳＭＣ的ＤＮＡ合成和细胞增殖，其作用呈剂量依赖性。雌孕激素联用比单用孕激素效果明显，合成的孕激素左旋十八甲基炔诺酮（ＬＮＧ）和３－酮－地索高炔酮（ＤＳＧ）促进ＨＵＡＳＭＣ细胞增殖作用弱于天然际酮。睾酮对ＨＵＡＳ     

TGFβ1，bFGF对前列腺基质细胞增殖和分化的影响

目的　探讨前列腺基质增生的机制。　方法　系用ＭＴＴ和ＴＵＮＥＬ法检测体外原代无血清培养的前列腺基质细胞的增殖,用免疫组化、ＲＴＰＣＲ 方法检测平滑肌细胞特异性标记蛋白的表达。　结果　ＴＧＦβ有多重效应：浓度为０－０１ ～１０．００ｎｇ／ｍｌ 的ＴＧＦβ可抑制指数增生期前列腺基质细胞增殖;浓度低于０－１ｎｇ／ｍｌ 的ＴＧＦβ刺激平顶期前列腺基质细胞增殖;浓度高于１ｎｇ／ｍｌ 的ＴＧＦβ促增殖平顶期的成纤维细胞向平滑肌细胞分化。ｂＦＧＦ对于指数增生期和平顶期细胞均有刺激增殖的作用,并抑制增殖平顶期的成纤维细胞向平滑肌细胞分化。　结论　ＴＧＦβ１／ｂＦＧＦ比例的改变有调节前列腺基质细胞的稳态平衡的作用。     

环境污染物三丁基氯化锡和氯化三苯锡对大鼠睾丸Leydig细胞的影响

目的:探讨环境污染物三丁基氯化锡(TBT)和氯化三苯锡(TPT)对大鼠睾丸Leyd ig细胞的影响。方法:①用0~80 nmol/L浓度的TBT和TPT处理大鼠睾丸Leyd ig(LC-540)细胞24~96 h,用四唑蓝(MTT)法确定细胞的存活率;②用DNA片段法确定是否存在细胞凋亡;③观察细胞内Ca2+螯合剂氨基苯乙烷四乙酸(BAPTA)和蛋白激酶A(PKA)抑制剂H-89、蛋白激酶C(PKC)抑制剂GF109203X、酪氨酸蛋白激酶(TPK)抑制剂Gen iste in是否可以阻断TBT所致的细胞凋亡;④检测TBT处理的原代大鼠睾丸Leyd ig细胞分泌睾酮的变化。结果:①TBT和TPT影响Leyd ig细胞存活率的强度基本相同,在20~80 nmol/L浓度时细胞存活率呈剂量和时间依赖性降低;②DNA片段法研究证实TBT和TPT可引起细胞凋亡;③BAPTA可阻断20 nmol/L的TBT所致的细胞凋亡,而PKA、PKC和TPK抑制剂对细胞存活率没有影响;④TBT可减少Leyd ig细胞分泌睾酮,并使其对人绒毛膜促性腺激素(hCG)刺激的反应性降低。结论:环境污染物TBT和TPT能直接导致睾丸Leyd ig细胞凋亡,并抑制睾酮分泌,这种致凋亡作用可能与细胞内Ca2+浓度增加有关。     

Effect of Typha capensis (Rohrb.)N.E.Br. rhizome extract F1 fraction on cell viability,apoptosis induction and testosterone production in TM3‐Leydig cells

Typha capensis (Rohrb.)N.E.Br. (bulrush) is used by traditional healers in Southern Africa to treat male reproductive problems. This study aimed at investigating the effects of T. capensis on TM3‐Leydig cells. T. capensis rhizome crude extract obtained from autumn, winter, spring and summer harvest was fractionated using HPLC into four fractions, and TM3‐Leydig cells were incubated with different concentrations of the F1 fraction (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) for 24, 48 and 96 hr respectively. The following parameters were evaluated: cell morphology, viability (MTT assay), testosterone production (testosterone ELISA test), apoptosis (Annexin V‐Cy3 binding) and DNA fragmentation (TUNEL assay). Results revealed that the summer harvest obtained the highest amount of extract. The F1 fraction of all harvests was the most effective. This fraction significantly enhanced testosterone production in TM3 cells in a dose‐dependent manner with maximum effect at 0.1 μg/ml. At higher concentrations, lower testosterone production was observed. Cell viability including apoptosis was not affected at concentrations used by the traditional healers to treat patients. This study shows that T. capensis enhanced testosterone production and might be useful to treat male infertility and ageing male problems.     

邻苯二甲酸二乙基己酯对小鼠胚胎Leydig细胞毒性作用研究

目的探讨邻苯二甲酸二乙基己酯(DEHP)对小鼠胚胎睾丸问质细胞(Leydig细胞)的毒性作用。方法小鼠胚胎Leydig细胞体外原代培养。观察DEHP导致的Leydig细胞形态及超微结构改变,用MTT法和Annexin-V／PI流式细胞术分别检测DEHP在培养基内终浓度为25、510、100和200mg／L时对Leydig细胞活力影响及毒性作用。结果Leydig细胞活力随着DEHP浓度增加和作用时间延长而明显下降。Annexin-V／PI流式细胞术检测提示:死亡细胞比例随DEHP浓度升高和作用时间延长而显著增加,同时细胞增殖速度变慢,甚至增殖停止,伴随一系列细胞形态学及超微结构改变,呈剂量、时间依赖关系。结论DEHP能降低Leydig细胞的活性并促进其死亡,有明显细胞毒性。     

青蒿素联合全转铁蛋白对肝癌的选择性抑制作用

目的 观察青蒿素联合全转铁蛋白在体外对人肝癌SMMC-7721细胞的选择性抑制作用.方法 采用嚷唑蓝(MTT)着色细胞计数及锥虫蓝染色计数检测青蒿素联合全转铁蛋白对人肝癌SMMC-7721细胞的细胞生长、增殖和细胞活力的影响.结果 青蒿素在各种浓度显著地抑制SMMC-7721细胞的生长、集落形成以及细胞活力(P<0.05).青蒿素导致所有实验细胞株的肿瘤细胞死亡,但是敏感性有所变化,在加入全转铁蛋白后,青蒿素对人肝癌SMMC-7721细胞的选择性毒性作用明显增强(P<0.01).结论 青蒿素联合全转铁蛋白能有效地抑制人肝癌SMMC-7721细胞的生长、细胞增殖和细胞活力.     

Regulation of testosterone production in the adjuvant-induced arthritic rat.

Serum testosterone concentrations are reduced in men with rheumatoid arthritis and in rats with adjuvant-induced arthritis, a common model for rheumatoid arthritis. To understand the mechanism responsible for this reduction, testosterone production by testicular cells and Percoll-purified Leydig cells from nonarthritic and arthritic rats was studied. Leydig cells in crude interstitial cell preparations from arthritic rats secreted significantly less testosterone in response to human chorionic gonadotropin (hCG) stimulation than cells from nonarthritic animals. In contrast, no differences in hCG and dibutyryl cyclic adenosine monophosphate-stimulated testosterone production by Percoll-enriched Leydig cells from arthritic and nonarthritic animals were observed. To determine whether a secretory product from testicular macrophages was important to this reduction, macrophages from arthritic and nonarthritic animals were cultured. The conditioned media from these cultures were added to cultures of interstitial cells from nonarthritic animals. Nonarthritic rat testicular macrophage-conditioned medium had no significant effect on testosterone production. In contrast, conditioned medium from arthritic rat testicular macrophages significantly reduced testosterone production. These results suggest that testicular macrophages secrete a factor that may be important in the regulation of testosterone production in the adjuvant-induced arthritic rat.     

Leydig cell cooperation in vitro: evidence for communication between adult rat Leydig cells

In short-term incubations (32 C, 3 h) of purified adult rat Leydig cells, increasing the density from 5000 to 50,000 cells/16 mm diameter culture well caused a significant increase in human chorionic gonadotropin (hCG)-stimulated testosterone secretion/cell. Density-dependent stimulation was also observed under basal conditions and in the presence of dibutyryl cyclic adenosine monophosphate, luteinizing hormone-releasing hormone, or 22-hydroxy cholesterol. In contrast, increasing the incubation density of purified Leydig cells by addition of other testicular cells had no effect on basal or hCG-stimulated testosterone secretion. hCG-stimulated testosterone secretion by Leydig cells incubated at low density was also increased by addition of Leydig cell-conditioned medium. This stimulatory activity was removed by charcoal extraction and by ultrafiltration (approximately 30 kDa cut-off). The data indicate that Leydig cells cooperate by secretion of low molecular weight, cell-specific stimulatory factors that support Leydig cell steroidogenesis in vitro, and may also play a role in regulating Leydig cell function in vivo.     

Taurine ameliorates cytotoxic effects of Di(2-ethylhexyl) phthalate on Leydig cells

It has been revealed that di(2-ethylhexyl)phthalate (DEHP) has toxic impacts on the male reproductive system. Taurine (TAU) is an amino acid with antioxidant property and beneficial impacts on the male reproductive system. In this study, protective impacts of Taurine (TAU) on DEHP-induced Leydig TM3 cell toxicity were investigated. The cells exposed to DEHP (0.8 µmol) or TAU (100 mg/ml) for 24 hr. Cell viability (MTT assay), apoptosis, oxidative stress and testosterone level were examined. DEHP could significantly decrease the cell viability percentage, reduce testosterone level, increase apoptosis, elevate Bax/ Bcl-2 ratio and enhance caspase-3 and -9 activity in the TM3 cells. Additionally, DEHP significantly elevated malondialdehyde contents and reactive oxygen species levels. It also augmented superoxide dismutase and catalase activity in the Leydig cells. Co-treatment of DEHP with TAU increased viability and testosterone level, while oxidative stress and apoptosis significantly reduced. TAU could decrease Bax/Bcl-2 ratio and caspase-3 and -9 activity in the DEHP-intoxicated cells. Our results have clearly shown that TAU protects TM3 cells against oxidative stress and apoptosis induced by DEHP.     

大鼠睾丸Leydig细胞体外增殖研究

目的:探讨通过优化细胞培养体系,实现Leydig细胞的体外增殖培养。方法:联合应用胶原酶消化、不锈钢滤网过滤及差速贴壁法获得3周龄雄性Wistar大鼠睾丸Leydig细胞,贴壁细胞以DMEM/F12培养液及优化培养体系培养,MTT法、细胞计数法检测培养细胞的增殖能力;分别对原代培养2h、4d细胞进行3β-HSD免疫化学染色及流式细胞术分析,检测细胞成分,同时检测培养细胞睾酮在hCG刺激下分泌能力变化。结果:优化培养体系能够明显促进3周龄大鼠睾丸Leydig细胞大量增殖,群体倍增时间为(2.26±0.31)d,传统培养体系培养细胞群体倍增时间为(16.32±2.14)d,两者差异有显著性(P<0.05);原代细胞经流式细胞术鉴定,3β-HSD阳性细胞所占比例分别为(54.3±7.1)%,培养4d后,增殖细胞3β-HSD阳性细胞率为(93.6±4.6)%。增殖细胞均有睾酮生成功能,在hCG刺激下睾酮分泌均明显上升(P<0.05)。结论:优化培养体系能够促进差速贴壁法获得的睾丸Leydig细胞大量增殖。     

Morphological and Endocrinological Differences between the Abdominal Testes in Bilateral and Unilateral Cryptorchid Rats

Using a new experimental model of cryptorchism in rats, where testicular descent was prevented, testicular development and function were studied in bilateral and unilateral cryptorchid animals Morphometric and radioimmunological techniques were used. Up to 30 days after birth testicular development was identical in the two types of abdominal testes but in adult rats differences were observed. In these rats spermatogenesis was damaged to a similar extent, but total tubular length and testicular weight were increased in the bilateral abdominal testes. Moreover, in these testes, the volume density of Leydig cells, the total Leydig cell mass, the average Leydig cell size and the testis testosterone concentration were larger than in unilateral abdominal testes. It is suggested that the impaired spermatogenesis seen in both kinds of abdominal testes may be unrelated to Leydig cell function.