BDNF基因修饰神经干细胞移植对大鼠脊髓损伤后神经细胞凋亡的影响

采用电控大鼠脊髓损伤打击装置致大鼠脊髓损伤模型。将120只SD大鼠随机分为假手术组（Sham组）、脊髓损伤组（SCI组）、神经干细胞组（NSC组）、BDNF基因修饰神经干细胞组（NSC—BDNF组）。免疫组织化学法检测各组脊髓BDNF、Bax、Bcl-2的表达，流式细胞仪检测脊髓细胞凋亡率。结果 NSC—BDNF组BDNF免疫阳性细胞光密度值较NSC、SCI组明显增加（P〈0．05），表达时间及高峰延长；且Bcl-2蛋白表达较其他组在各时点均增高（P均〈0．05）。而Bax蛋白表达较其他组在各时点均降低（P均〈0．01）。凋亡率亦降低（P均〈0．01）。提示BDNF基因修饰NSC可在损伤脊髓内有效表达。且明显促进脊髓损伤后Bcl-2的高表达，抑制Bax的表达，从而降低神经细胞的凋亡率。     

BDNF基因修饰神经干细胞移植对脊髓损伤后红核神经元作用的实验研究

将20只SD大鼠随机分为假手术组、脊髓损伤组（SCI组），神经干细胞（NSC）组（NSC组）及脑源性神经营养因子（BDNF）基因修饰NSC组（BDNF组）各5只。假手术组仅行椎板切开术；余三组采用电控脊髓损伤打击装置致脊髓损伤模型，术后3d SCI组脊髓内注射生理盐水溶液5μl；NSC组注射NSC悬液5μl，BDNF组注射NSC-BDNF悬液5μl。术后观察各组行为学（BBB）评分和红核神经元存活情况。结果BDNF组BBB评分显著高于SCI组、NSC组．红核神经元数目均多于SCI组、NSC组（P均〈0．01）。提示BDNF基因修饰NSC移植对脊髓损伤后红核神经元有保护作用。     

氟对小鼠成骨细胞增殖的影响

目的 观察氟对体外培养小鼠成骨细胞增殖的影响。方法 原代培养小鼠成骨细胞，以0（对照组）、5、10、20、40mg／L剂量染氟，光、电镜观察成骨细胞的形态学变化；采用生长曲线、噻唑蓝（MTT）法及流式细胞仪检测细胞周期，观察细胞数量变化和细胞周期时相分布。结果 第10天时，5、10、20mg／L染氟组从形态学上表现出细胞增殖。而40mg／L组中出现细胞凋亡。染氟24h时，20mg／L组与对照组相比细胞增殖明显，差异具有统计学意义（P〈0．05），随着时间延长，染氟组表现出更强的增殖活性，至72h时，各染氟组成骨细胞增殖均高于对照组（P〈0．05）。24h时各染氟组间细胞增殖水平未见明显差异（P〉0．05），48h以后40mg／L组明显低于其他各染氟组（P〈0．05）。而染氟72h时5mg／L组增殖强度也低于10、20mg／L组（P〈0．05）。与对照组相比，10、20mg／L组处于G0／G1期的细胞数减少，而S期细胞数增加（P〈0．05），同时两组的细胞增殖指数也高于对照组（P〈0．05），生长表现出增殖状态；其中10mg／L组与其他染氟组之间，差异有统计学意义（P〈0．05），表现为细胞增殖能力增强。结论 低剂量的氟在较短作用时间内可以促进体外培养小鼠成骨细胞增殖，随剂量增加和暴露时间延长其促进作用减弱。     

神经干细胞与神经营养因子

神经干细胞（NSC）增殖是神经发育早期阶段的主要事件，在此阶段，NSC通过10—12次分裂进行增殖，为后期神经发生提供足够的细胞来源。此外，当脑组织受损时，内源性NSC也可增殖并分化，补偿缺失的神经细胞，部分修复神经功能。神经营养因子（NTF）是一类对神经元有特异性保护作用的内分泌多肽，它可促进体内、外培养神经元存活及突起生长。1952年Levi—Montalcini在研究鸡胚的神经发育过程中发现了神经生长因子（NGF），此后几十年，新的NTF不断被发现，并形成了NTF大家族。它们来源于靶细胞逆向营养神经元，促进和维持神经细胞生长、存活，修复神经细胞功能。在胚胎发育早期，NTF如碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）等能显著促进NSC的增殖与分化。     

脂蛋白(a)及高敏C-反应蛋白与冠状动脉狭窄病变的相关研究

目的探讨脂蛋白（a）[Lp（a）]、高敏C-反应蛋白（hs—CRP）与冠状动脉（冠脉）狭窄的相关性。方法480例怀疑患有冠心病的患者在冠状动脉造影术前测定血清LD（a）和hs—CRP，冠脉病变的范围用冠脉病变的血管支数表示。结果①冠脉狭窄病变组351例，对照组129例。冠脉狭窄病变组患者的血清Lp（a）和hs—CRP水平分别为（129．18±2．21）mg／L和（1．68±3．40）mg／L，对照组分别为（50．18±1．84）mg／L和（0．81±2．86）mg／L，冠脉狭窄病变组患者的血清Lp（a）和hs—CRP水平均显著高于对照组（P〈0．01）。②单支病变组133例，双支病变组118例，三支病变组100例。单支病变组Lp（a）和hs—CRP水平分别为（116．90±2．15）mg／L和（1．54±3．24）mg／L，双支病变组分别为（128．77±2．16）mg／L和（1．89±3．14）mg／L，三支病变组分别为（148．15±2．31）mg／L和（1．99±3．95）mg／L。单支、双支及三支病变组LD（a）及hs—CRP均显著高于对照组（P〈0．01）。③Logistic回归分析结果显示，Lp（a）〉300mg／L者，发生冠心病的危险性增加（OR为1．025，95％CI：1．018～1．032，P〈0．001）；hs—CRP〉3．00mg／L者，发生冠心病的危险性增加（OR为1．281，95％CI：1．120～1．465，P〈0．001）。④Lp（a）与hs—CRP之间呈正相关（r=0．172，P〈0．01）。结论血清Lp（a）和hs—CRP水平与冠脉狭窄病变密切相关，均是冠心病的独立危险因素。     

红霉素对铜绿假单胞菌蹭行运动的抑制

目的体外培养铜绿假单胞菌（pseudomonasaeruginosa，PA），观察低浓度红霉素对铜绿假单胞菌蹭行运动（twitchingmotility）的影响。方法将铜绿假单胞菌ATCC1244菌株接种于3组不同浓度红霉素（2．5mg／L、0.5mg／L、0.25ms／L）的细菌培养基中，同时接种于未加红霉素的对照培养基中，采用肉眼观察、免疫荧光显微镜检查、免疫印迹实验、点杂交实验、电镜观察等方法观察铜绿假单胞菌蹭行运动的变化及鉴别其蹭行运动的分子组成。结果经过18—36h的孵育，LB平板上不同浓度的红霉素对PA蹭行运动有抑制作用，细菌光晕（halation）直径与药物浓度呈反比；18h时细菌的平均光晕直径2．5mg／L组为（0.48±0．14）cm，0．5mg／L组为（0．64±0．20）cm，0．25mg／L组为（0.95±0.18）cm，对照组为（1．40±0．21）cm，组间比较差异有统计学意义（F=123．15，P〈0．01）；24h时2．5mg／L组为（0．67±0．12）cm，0．5mg／L组为（0．82±0.23）cm，0．25mg／L组为（1．18±0．24）cm，对照组为（1．58±0．28）cm，组间比较差异有统计学意义（F=76．37，P〈0．01）；36h时2．5mg／L组为（0.91±0．17）cm，0．5mg／L组为（1．04±0．32）cm，0．25mg／L组为（1．49±0．31）cm，对照组为（2．07±0．38）cm，组间比较差异有统计学意义（F=54．75，P〈0．01）。免疫荧光显微镜观察结果可见细菌菌体一端呈簇状突起，为Ⅳ型菌毛的位置所在，簇状突起的多少与药物浓度呈反比。免疫印迹实验结果显示，随着药物浓度的减少，Ⅳ型菌毛的主要成分PilA蛋白表达增加。点杂交实验结果显示在蹭行运动中起作用的PilA蛋白主要表达在光晕的最外周，但组间未见明显区别。电镜下可见红霉素作用后PA菌体一端的Ⅳ型菌毛较对照组减少，部分脱离菌体。结论低浓度红霉素对PA的蹭行运动有抑制作用，且与红霉素浓度呈正比。     

两种剂量阿托伐他汀对冠心病患者踝臂指数和hs-CRP影响的比较

目的：观察不同剂量阿托伐他汀治疗冠心病的疗效及其对踝臂指数（ABI）和高敏C反应蛋白（hs-CRP）的影响。方法：选择冠心病患者110例,随机分为40mg组和10mg组,每组各55例。10mg组予以阿托伐他汀10mg／次,1次／晚,40mg组予以阿托伐他汀40mg／次,1次／晚。观察比较两组的血脂及治疗前后的ABI和hs-CRP的水平。结果：治疗后,两组的总胆固醇（TC）,三酰甘油（TG）和低密度脂蛋白胆固醇（LDL-C）的水平均较治疗前显著降低（P〈0．05或P〈0．01）,高密度脂蛋白胆固醇（HDL-C）水平较治疗前显著升高（P〈0．05）;与10mg组比较,40mg组LDL,C较10mg组降低更显著（P〈0．05）。hs-CRP水平较治疗前也显著降低（P〈0．01）;与10mg组比较,40mg组hs-CRP水平下降更为显著（P〈0．05）。而ABI水平治疗后较治疗前显著升高（P〈0．01）,与lOmg组比较,40mg组ABI的改善更为显著（P〈0．05）。结论：阿托伐他汀对冠心病患者的ABI和hs-CRP改善明显,而且40mg组改善更显著。     

糖尿病大鼠胃肠动力及肌间神经丛形态学改变

目的 了解糖尿病大鼠胃肠动力障碍时，肌间神经丛有无形态学异常并探讨胃肠功能异常的机制。方法 30只大鼠分成对照组（10只）和糖尿病组（20只），糖尿病组用链脲佐菌素建立大鼠糖尿病模型，4个月后测定两组大鼠胃肠传输速率，并用酶组织化学方法观察回肠肌间神经丛内胆碱能和氮能神经的组织学改变。结果 糖尿病组大鼠胃肠传输速率明显延迟，回肠肌间神经丛内胆碱能神经元密度明显降低（P〈0．01），氮能神经节和氮能神经元的密度均显著升高（P〈0．05和P〈0．01）。结论 糖尿病大鼠小肠肌间神经丛内胆碱能神经减少，氮能神经增多，这可能是导致胃肠传输速率延迟的重要原因，由此引起小肠运动障碍。     

普伐他汀对急性冠状动脉综合征患者血浆sCD40L及MMP-9影响的观察

选择急性冠状动脉综合征（ACS）患者35例（ACS组），稳定型心绞痛（SAP）患者31例（SAP组），正常健康体检者28例（／E常对照组），前两组口服普伐他汀20mg／d，连用8周。采用酶联免疫吸附法（ELISA）测定三组治疗前后血浆可溶性白细胞分化抗原40配体（sCD40L）及基质金属蛋白酶-9（MMP-9）浓度。结果治疗前ACS组血浆sCD40L及MMP-9显著高于SAP组及正常对照组（P〈0．05），SAP组MMP-9显著高于正常对照组（P〈0．05），SAP组sCD40L与正常对照组之间无差异（P〉0．05），普伐他汀治疗8周后ACS组血浆sCD40L及MMP-9水平显著下降（P〈0．05，P〈0．01），SAP组MMP-9显著下降（P〈0．05），sCD40L无明显改变（P〉0．05）。认为sCD40L及MMP-9升高可能与ACS发病机制密切相关，sCD40L及MMP-9可作为ACS诊断及病情判断的重要指标；普伐他汀能降低ACS患者sCD40L及MMP-9水平。     

间歇性和持续性哮喘患者呼出气冷凝液中的白三烯C4、8-异前列腺素与硝酸盐检测的临床意义

目的探讨支气管哮喘（简称哮喘）患者治疗前后呼出气冷凝液（EBC）中白三烯c4（LTC4）、8-异前列腺素（8-ISO）、硝酸盐（NO。）水平及临床意义。方法酶法和比色法对30例哮喘患者EBC中LTC4、8-ISO、NO。进行检测,30例健康者作为对照。结果哮喘患者的LTC4比对照组显著升高（55．17ng／L比17．95ng／L,P〈0．01）,持续期比间歇期组显著升高（67．38ng／L比41．21ng／L,P〈0．01）,持续期比对照组显著升高（67．38ng／L比17．95ng／L,P〈0．01）,间歇期比对照组显著升高（41．21ng／L比17．95ng／L,P〈0．01）,患者组比治疗后组显著升高（55．17ng／L比38．36ng／L,P〈0．01）。哮喘患者的8-ISO比对照组显著升高（13．41ng／L比6．93ng／L,P〈0．01）,持续期组比对照组显著升高（14．29ng／L比6．93ng／L,P〈0．01）,间歇期比对照组显著升高（12．40ng／L比6．93ng／L,P〈0．05）。哮喘患者的NOx比对照组显著升高（4．17ng／L比3．20ng／L,P〈0．01）,持续期组比对照组显著升高（4．50ng／L比3．20ng／L,P〈0．01）。结论LTC4、8-ISO、NOx水平能够很好的体现出哮喘患者的气道炎症水平,而孟鲁斯特药物能够很好的控制气道炎症水平,缓解气道炎症。     

消化系恶性肿瘤病人LAK细胞和NK细胞功能与表型的变化

通过观察20例正常人和24例消化系恶性肿瘤病人外周血自然杀伤细胞(NK)和淋巴因子激活的杀伤细胞(LAK)的活性变化,以及加用重组白细胞介素2(rIL-2)刺激前后T淋巴细胞表型变化。结果发现肿瘤病人的NK细胞活性明显下降,但经rIL-2激活后LAK细胞活性得到明显提高,其溶解率接近正常水平。肿瘤病人的总T淋巴细胞(CD_(3+))和辅助/诱导T淋巴细胞(CD_(4+))水平低于正常,但抑制/杀伤淋巴细胞(CD_(8+))水平正常。辅助/诱导淋巴细胞与抑制/杀伤淋巴细胞之比为1.18,低于正常水平(1.55)。经加入rIL-2培养后,CD_(3+)和CD_(8+)淋巴细胞的比率明显升高并达正常水平。而在正常人此变化不明显,且加用rIL-2培养与不加者无显著差异。IL-2受体的表达正常人与肿瘤病人无异。结果显示胃肠道恶性肿瘤病人的免疫机制受到抑制,但能被IL-2提高至正常水平。     

Robust measurement of telomere length in single cells

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.Telomeres are the ribonucleoprotein structures that cap and protect linear chromosome ends from genomic instability and tumorigenesis (1, 2). Intriguingly, telomere shortening protects against tumorigenesis by limiting cell growth (3, 4), but also can impair tissue regenerative capability and cell viability (5, 6).Thus far, most assays of telomere length measure average telomere length from aggregates of many cells derived from dissected tissues, cultured cells, or blood (7). Telomere restriction fragment (TRF) determination (1, 8), a Southern blot-based technique, remains the “gold standard” for determining absolute telomere length, but requires a large amount of starting material (0.5–5 µg DNA) and several days for processing. Moreover, the requirements for gel electrophoresis and hybridization limit the scalability of this assay. Recently, a quantitative PCR (qPCR)-based method for telomere length measurement was developed, providing the convenience and scalability of PCR (9). Although the DNA requirement (35 ng) for qPCR is significantly less than TRF, it still relies on populations of cells to derive sufficient amount of DNA.Quantitative FISH (Q-FISH) allows sensitive visualization of relative telomere length from individual cells and individual telomeres, but this method requires many cells or metaphase arrested cells, which precludes its application to many sample types, including postmitotic cells, senescent cells, and other nondividing cells, and when only one actual cell is required to test. In addition, preparing chromosome spreads requires significant technical skill, and only proliferating cells within a population reach metaphase stage, so this analysis potentially biases the estimates of telomere length for a given cell population (10–12). High-throughput Q-FISH, flow FISH, and single telomere length analysis can be used for telomere measurement of dividing, nondividing, and senescent cells, but these methods also require large cell populations (13–15).The ability to measure telomere length in single cells rather than relying upon average telomere length in cell populations or the entire tissue enables the study of biological heterogeneity on a cell-by-cell basis, an issue of fundamental importance for studies of aging, development, carcinogenesis, and many other diseases. Here, we demonstrate an accurate determination of telomere length in individual cells, with the resolution and scalability of the qPCR telomere length assay.The basis of qPCR is that within a given cell, the ratio of the copy number of telomere repeats to the copy number of a multicopy reference gene is fixed (3), and this method, because of its simplicity, has been widely used to investigate a variety of telomere shortening-associated diseases (7), even sensitive enough to identify mild telomere dysfunction resulting from chronological life stress (16, 17). We adapted qPCR to measure telomere length in individual cells by using a preamplification step that specifically targets both the telomere and multicopy genes, followed by a qPCR assay to obtain telomere to reference gene (T/R) ratio. A single-cell telomere (SCT) length measurement method (SCT-pqPCR) runs robustly, and shows an identical T/R ratio for two sister blastomeres from two-cell–stage mouse embryos. The average result from SCT-qPCR with multiple single cells is linearly correlated to Q-FISH, TRF, and conventional qPCR assays designed for a large number of cells. The heterogeneity of telomere length among several populations of cells by SCT-pqPCR run on multiple single cells is consistent with—and sometimes superior to—results obtained by Q-FISH. Application of SCT-pqPCR to study telomere length during early embryo development, aging, and cancer demonstrate the value of this single-cell telomere length assay method.     

Glycolipid presentation to natural killer T cells differs in an organ-dependent fashion

It has been shown that dendritic cells (DCs) are able to present glycolipids to natural killer (NK) T cells in vivo. However, the essential role of DCs, as well as the role of other cells in glycolipid presentation, is unknown. Here, we show that DCs are the crucial antigen-presenting cells (APCs) for splenic NK T cells, whereas Kupffer cells are the key APCs for hepatic NK T cells. Both cell types stimulate cytokine production by NK T cells within 2 h of glycolipid administration, but only DCs are involved in the systemic, downstream responses to glycolipid administration. More specifically, CD8alpha+ DCs produce IL-12 in response to glycolipid presentation, which stimulates secondary IFN-gamma production by NK cells in different organs. Different APCs participate in glycolipid presentation to NK T cells in vivo but differ in their involvement in the overall glycolipid response.     

Processivity of peptidoglycan synthesis provides a built-in mechanism for the robustness of straight-rod cell morphology

The propagation of cell shape across generations is remarkably robust in most bacteria. Even when deformations are acquired, growing cells progressively recover their original shape once the deforming factors are eliminated. For instance, straight-rod-shaped bacteria grow curved when confined to circular microchambers, but straighten in a growth-dependent fashion when released. Bacterial cell shape is maintained by the peptidoglycan (PG) cell wall, a giant macromolecule of glycan strands that are synthesized by processive enzymes and cross-linked by peptide chains. Changes in cell geometry require modifying the PG and therefore depend directly on the molecular-scale properties of PG structure and synthesis. Using a mathematical model we quantify the straightening of curved Caulobacter crescentus cells after disruption of the cell-curving crescentin structure. We observe that cells straighten at a rate that is about half (57%) the cell growth rate. Next we show that in the absence of other effects there exists a mathematical relationship between the rate of cell straightening and the processivity of PG synthesis—the number of subunits incorporated before termination of synthesis. From the measured rate of cell straightening this relationship predicts processivity values that are in good agreement with our estimates from published data. Finally, we consider the possible role of three other mechanisms in cell straightening. We conclude that regardless of the involvement of other factors, intrinsic properties of PG processivity provide a robust mechanism for cell straightening that is hardwired to the cell wall synthesis machinery.     

Regenerative medicine for the treatment of heart disease

Heart failure is a major cause of mortality worldwide with a steady increase in prevalence. There is currently no available cure beyond orthotopic heart transplantation, which for a number of reasons is an option only for a small fraction of all patients. Considerable hope has therefore been placed on the possibility of treating a failing heart by replacing lost cardiomyocytes, either through transplantation of various types of stem cells or by boosting endogenous regenerative mechanisms in the heart. Here, we review the current status of stem and progenitor cell‐based therapies for heart disease. We discuss the pros and cons of different stem and progenitor cell types that can be considered for transplantation and describe recent advances in the understanding of how cardiomyocytes normally differentiate and how these cells can be generated from more immature cells ex vivo. Finally, we consider the possibility of activation of endogenous stem and progenitor cells to treat heart failure.     

Sickle cell vasoocclusion: heterotypic, multicellular aggregations driven by leukocyte adhesion

Homozygous expression of sickle beta-globin alters the function of blood cells and the endothelium, producing a wide spectrum of clinical manifestations. Intravital microscopy studies in sickle cell mice suggest that vasoocclusion is a complex, sequential, multistep phenomenon involving (1) endothelial activation by sickle erythrocyte (SSRBC), (2) leukocyte (WBC) adhesion to the endothelium, and (3) the direct interaction between SSRBCs and adherent WBCs, which leads to reduced blood flow and tissue ischemia. Each of these steps represents a potentially useful therapeutic target. The identification of molecular determinants mediating vasoocclusion will provide new strategies for the prevention and treatment of this debilitating illness.     

特应性皮炎的免疫学发病机制

特应性皮炎是慢性复发性炎症性皮肤疾病，发病机制复杂，其中变态反应因素在发病机制中扮演着重要角色。目前认为Th1／Th2平衡失调是特应性皮炎重要的发病机制。本文围绕这一机制综述T细胞、树突状细胞、角质形成细胞及IgE在特应性皮炎发病机制中的作用。     

Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry

Left-right (LR) asymmetry (handedness, chirality) is a well-conserved biological property of critical importance to normal development. Changes in orientation of the LR axis due to genetic or environmental factors can lead to malformations and disease. While the LR asymmetry of organs and whole organisms has been extensively studied, little is known about the LR asymmetry at cellular and multicellular levels. Here we show that the cultivation of cell populations on micropatterns with defined boundaries reveals intrinsic cell chirality that can be readily determined by image analysis of cell alignment and directional motion. By patterning 11 different types of cells on ring-shaped micropatterns of various sizes, we found that each cell type exhibited definite LR asymmetry (p value down to 10(-185)) that was different between normal and cancer cells of the same type, and not dependent on surface chemistry, protein coating, or the orientation of the gravitational field. Interestingly, drugs interfering with actin but not microtubule function reversed the LR asymmetry in some cell types. Our results show that micropatterned cell populations exhibit phenotype-specific LR asymmetry that is dependent on the functionality of the actin cytoskeleton. We propose that micropatterning could potentially be used as an effective in vitro tool to study the initiation of LR asymmetry in cell populations, to diagnose disease, and to study factors involved with birth defects in laterality.     

Identification of an inhibitory factor from a Sertoli clonal cell line (TM4) that modulates adult rat Leydig cell steroidogenesis

Sertoli cell produces several biological factors that modulate Leydig cell steroidogenic function by either stimulating or inhibiting its testosterone production. We have evaluated the effect of an inhibitory factor in the spent media of a Sertoli clonal cell line (TM4) which inhibits Leydig cell steroidogenesis. The presence of such an inhibitory factor in TM4 media was bioassayed using Percoll purified Leydig cells isolated from adult rats with purity of greater than 95%. TM4 media inhibited both human chorionic gonadotropin (hCG)-stimulated testosterone and cAMP production by purified Leydig cells dose-dependently but had no apparent effect on 8-bromo-cAMP- and forskolin-stimulated testosterone production. Also it did not interfere with the binding of [¹²⁵I]hCG to Leydig cells. TM4 media inhibited cholera toxin-stimulated testosterone production as well as forskolin- and cholera toxin-stimulated cAMP production. The mechanism of action of this factor in TM4 media appears to be different from transforming growth factor (TGF-β) which inhibited both 8-bromo-cAMP- and forskolin-stimulated testosterone production and inhibited the binding of [¹²⁵I]hCG binding to Leydig cells. The inhibitory factor contained in TM4 media has been partially purified by sequential preparative anion exchange and C-18 reversed-phase high-performance liquid chromatography. In summary, the Sertoli TM4 cell line produces at least one potent inhibitory factor which decreases the responsiveness of purified Leydig cells to hCG stimulation with a dramatically different mechanism from other currently known Leydig cell inhibitory factors; this protein may serve as a valuable tool to study testicular paracrine regulation.