TRH受体在大鼠睾丸内的表达及其在间质细胞中的作用

发布时间：2018-06-26 19:53

本文选题：促甲状腺激素释放激素 + 促甲状腺激素释放激素受体　；参考：《第四军医大学》2008年博士论文

【摘要】： 背景与目的促甲状腺激素释放激素(thyrotrophin-releasing hormone, TRH)最初是从羊和猪的下丘脑中提取并纯化的3肽,它与垂体前叶促甲状腺激素细胞表面特异性受体(thyrotrophin-releasing hormone receptors, TRHRs)结合,刺激促甲状腺激素(TSH)的合成和分泌。在哺乳动物体内先后克隆出了针对TRH的两个受体,TRH-R1和TRH-R2,两者同属G蛋白耦联受体家族成员,在同种动物中其氨基酸序列具有50%的一致性。具有免疫活性的TRH及TRH受体不仅分布于下丘脑及其以外的中枢神经系统内,还广泛存在于外周组织中。睾丸是外周组织中为数不多的可同时表达TRH及其两个受体的器官,但TRH及其受体在睾丸内的功能尚不清楚,因而本实验的目的在于探讨TRHRs在大鼠睾丸内表达的意义。研究方法和内容 1.利用蛋白质免疫印迹杂交及免疫组织化学方法对比研究出生后大鼠发育不同阶段(8,14,21,35,60和90天)睾丸中TRH-R2和TRH-R1的表达和定位,并结合图像分析技术对其表达进行半定量分析。 2.采用Percoll连续密度梯度法离心法提取、纯化并原代培养成年大鼠(90天)睾丸Leydig细胞,探讨不同剂量TRH对Leydig细胞睾酮合成的影响。 3.合成EDS,构建并验证EDS诱导的Leydig细胞凋亡模型;采用Western blotting、免疫组织化学以及免疫荧光双标的方法,观察在EDS处理后成年型Leydig细胞再生过程中TRH-R1和TRH-R2的表达情况。 4.从出生后21d大鼠睾丸内分离、纯化Leydig祖细胞,采用BrdU掺入实验,观察不同剂量的TRH对Leydig祖细胞DNA合成的影响。结果 1.Western blotting和免疫组织化学均检测到了TRH-R1和TRH-R2在大鼠睾丸出生后不同阶段的表达,定位在Leydig细胞,阳性物质位于胞浆和胞膜,同一个Leydig细胞可同时表达TRH-R1和TRH-R2。图像分析显示两者在不同阶段Leydig细胞内的表达强度有所不同,TRH-R2最大表达量为出生后35d睾丸,主要为新形成及未成熟的Leydig细胞,而TRH-R1则在成年大鼠睾丸内表达较高,主要为成熟的Leydig细胞。 2.Percoll连续密度梯度离心法提取成年大鼠睾丸Leydig细胞,经3β-HSD酶细胞化学染色,纯度符合细胞学实验要求。在给予不同剂量TRH处理后,原代培养的Leydig细胞睾酮分泌受到影响,并呈剂量依赖性。相对低剂量的TRH(0.01和0.1 ng/ml)可显著抑制ALCs基础睾酮和hCG刺激下的睾酮分泌,P值小于0.01;而相对高剂量的TRH(1和10 ng/ml)却促进了hCG刺激下的睾酮分泌(P0.05)。虽然1 ng/ml TRH组与0.1 ng/ml TRH在基础睾酮分泌上没有统计学差异,1 ng/ml TRH刺激下,ALCs睾酮水平亦有所升高。 3.按照75mg/kg的剂量一次性给予大鼠腹腔注射EDS后,睾丸Leydig细胞在2 d后可全部凋亡消失,经血清睾酮测量、睾丸恒冷箱切片3β-HSD酶细胞化学染色、石蜡切片HE染色以及3β-HSD的免疫组织化学染色等证实,动物模型复制成功。一次性腹腔注射EDS后,Leydig细胞在24hr内即发生退化,2-3天后,Leydig细胞全部消失。EDS处理后14d,在间质管周区出现长梭形Leydig细胞的再生,至处理后28d新生的Leydig细胞数量增加并由管周向间质中央迁移,变为多角形。在EDS处理后的14d,Western blotting和免疫组织化学染色检测到了TRH-R1和TRH-R2在睾丸内的表达,此时对应着大鼠睾丸Leydig祖细胞的再生,经利用荧光双标进一步证实,TRH-R1和TRH-R2阳性细胞即为再生的Leydig细胞。 4.采用Percoll连续密度梯度离心法从出生后14d大鼠中成功分离、纯化了Leydig祖细胞。在给予不同剂量的TRH(0.001-10 ng/ml)并向培养液中添加10μM BrdU后,PLCs的DNA合成可受到TRH的影响。在孵育12小时后,相对低剂量的TRH(0.001-0.1 ng/ml)可增加BrdU阳性标记细胞的数量和比例(从8.2%升高至11%以上),即PLCs的DNA合成增加,细胞的增殖或分化可能增多。但是相对高剂量的TRH(1 ng/ml)却不能促进PLCs的DNA合成,甚至BrdU掺入比例在10ng/ml TRH组略有下降(P=0.094)。将BrdU掺入时间延长至18和36hr,对照组和TRH处理组BrdU阳性细胞的数量和比例均增加(P0.01),即0.1ng/ml TRH可显著促进PLCs的DNA合成。结论 1.成功分离、纯化了大鼠睾丸PLCs和ALCs,可用于原代培养下细胞学实验。 2.成功合成EDS,并构建EDS诱导的成年大鼠Leydig细胞凋亡-再生模型。 3.TRH-R1和TRH-R2在大鼠睾丸出生后发育不同阶段以及EDS诱导的Leydig细胞凋亡模型中仅表达于Leydig细胞,即处于成年型Leydig细胞谱系不同阶段的Leydig细胞均表达TRH-R1和TRH-R2。另外,细胞培养显示,TRH能够影响成熟的ALCs睾酮合成分泌能力,以及Leydig祖细胞的DNA合成(增殖或分化)。故TRH及其受体参与了Leydig细胞谱系的发育和功能的调节。
[Abstract]:Background and purpose
Thyrotrophin-releasing hormone (TRH) is originally a 3 peptide extracted and purified from the hypothalamus of sheep and pigs. It combines with the thyrotrophin-releasing hormone receptors (TRHRs) receptor (thyrotrophin-releasing hormone receptors, TRHRs) in the anterior pituitary and stimulates the synthesis and secretion of thyroid stimulating hormone (TSH). Two receptors for TRH, TRH-R1 and TRH-R2, are cloned in the milk moving object, and both belong to the family members of the G protein coupling receptor family. The amino acid sequences of the same animals are consistent in the same animals. The immune active TRH and TRH receptors are distributed not only in the central nervous system of the hypothalamus and outside the hypothalamus, but also in the peripheral area. In the tissue, the testicles are the few organs in the peripheral tissue that can simultaneously express TRH and its two receptors, but the function of TRH and its receptors in the testis is not clear. The purpose of this experiment is to explore the significance of TRHRs expression in the testis of rats.
Research methods and contents
1. the expression and localization of TRH-R2 and TRH-R1 in the testis of the postnatal rats (8,14,21,35,60 and 90 days) were compared by the method of protein immunoblot hybridization and immunohistochemistry, and the expression was semi quantified by image analysis.
2. the Percoll continuous density gradient centrifugation was used to extract and purify the adult rat (90 days) rat testicular Leydig cells, and to explore the effect of different doses of TRH on the testosterone synthesis of Leydig cells.
3. EDS was synthesized and the EDS induced Leydig cell apoptosis model was constructed and verified. The expression of TRH-R1 and TRH-R2 during the regeneration of adult Leydig cells after EDS treatment was observed by Western blotting, immunohistochemistry and double immunofluorescence.
4. the Leydig progenitor cells were purified from the testis of 21d rats after birth, and the effects of different doses of TRH on the DNA synthesis of Leydig progenitor cells were observed by BrdU incorporation.
Result
1.Western blotting and immunohistochemistry detected the expression of TRH-R1 and TRH-R2 at different stages after the birth of the rat testis, located in the Leydig cells, the positive substance was located in the cytoplasm and the cytoplasm, and the same Leydig cell could simultaneously express the TRH-R1 and TRH-R2. image analysis to show that the expression intensity of the two in the different stages of Leydig cells in different stages. The largest expression of TRH-R2 is the postnatal 35d testis, mainly new and immature Leydig cells, while TRH-R1 is highly expressed in the testis of adult rats, mainly the mature Leydig cells.
2.Percoll continuous density gradient centrifugation was used to extract Leydig cells from adult rat testicles. After 3 beta -HSD enzyme cytochemical staining, the purity accords with the requirement of cytological experiment. After the treatment of different doses of TRH, the secretion of testosterone in the primary cultured Leydig cells is affected and is dose-dependent. The relative low dose of TRH (0.01 and 0.1 ng/ml) can be significantly inhibited. The testosterone secretion of ALCs based testosterone and hCG was less than 0.01, while the relative high dose of TRH (1 and 10 ng/ml) promoted the testosterone secretion under hCG stimulation (P0.05). Although the 1 ng/ml TRH group and 0.1 ng/ml TRH were not statistically different from the basal testosterone secretion, the level of testosterone was also elevated under 1 ng/ml.
3. after an intraperitoneal injection of EDS in rats at a dose of 75mg/kg, the apoptosis of Leydig cells in the testis disappeared after 2 D. By serum testosterone, 3 beta -HSD enzyme cytochemical staining, HE staining in paraffin section and immunohistochemical staining of 3 beta -HSD were confirmed by serum testosterone, and the animal model was replicated successfully. After EDS, Leydig cells degenerated within 24hr. After 2-3 days, all Leydig cells disappeared after.EDS treatment and 14d. The regeneration of long shuttle shaped Leydig cells appeared in the periinterstitial region. The number of Leydig cells in the newborn 28d increased and migrated from the peritubule to the mesenchyme, and turned into polygons.
The expression of TRH-R1 and TRH-R2 in the testis was detected by 14d, Western blotting and immunohistochemical staining after EDS treatment. At this time, the regeneration of Leydig progenitor cells in the rat testis was regenerated. It was further confirmed by double labeling fluorescence, that TRH-R1 and TRH-R2 positive cells were regenerated Leydig cells.
4. the Percoll continuous density gradient centrifugation was used to separate the Leydig progenitor cells from the postnatal 14d rats. After giving different doses of TRH (0.001-10 ng/ml) and adding 10 mu M BrdU to the culture medium, the PLCs DNA synthesis could be influenced by TRH. After 12 hours of incubation, the relatively low dose TRH could increase the positive rate. The number and proportion of the sex labeled cells (from 8.2% to more than 11%), that is, the DNA synthesis of PLCs increases, cell proliferation or differentiation may increase. But the relative high dose of TRH (1 ng/ml) does not promote DNA synthesis of PLCs, even the proportion of BrdU admixture decreases slightly in the 10ng/ml TRH group (P=0.094). The BrdU incorporation time is extended to 18 and 36hr, control, and 36hr, control The number and proportion of BrdU positive cells in both group and TRH treated group increased (P0.01), that is, 0.1ng/ml TRH significantly promoted DNA synthesis of PLCs.
conclusion
1. successfully isolated and purified PLCs and ALCs from rat testis, which can be used in primary culture cytology experiments.
2. EDS was successfully synthesized, and EDS induced apoptosis and regeneration model of adult rat Leydig cells was constructed.
3.TRH-R1 and TRH-R2 were expressed only in Leydig cells in the different stages of the development of rat testis and in the Leydig cell apoptosis induced by EDS, that is, the Leydig cells in the different stages of the adult Leydig cell lineage all express TRH-R1 and TRH-R2., and the cell culture shows that TRH can affect the capacity of the mature ALCs testosterone synthesis and secretion. And DNA synthesis (proliferation or differentiation) of Leydig progenitor cells. Therefore, TRH and its receptors are involved in the regulation of Leydig cell lineage development and function.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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