【作者】 李晓；

【导师】 陈洁；

【作者基本信息】 暨南大学 ， 泌尿外科（专业学位）， 2021， 硕士

【摘要】 目的:分析成纤维细胞生长因子对大鼠睾丸间质细胞增殖、分化及雄激素合成的调控,为了解成纤维细胞生长因子对睾丸间质细胞谱系的发育及治疗性腺功能减退等疾病提供初步的治疗依据。方法:利用Percoll密度梯度离心的方法,从出生后35天SD大鼠中分离出大鼠原代ILC,贴壁处理4小时后,用终浓度为10 ng/ml和100 ng/ml FGFs分别处理细胞48小时。利用高内涵显微镜分析ILCs细胞增殖情况并进行细胞计数;利用ELISA方法测各因子处理组培养基中上清雄激素含量。此外,利用相同的密度梯度方法,从出生90天的SD大鼠中分离出ALCs,用终浓度为10 ng/ml和100 ng/ml FGFs分别处理细胞24小时,ELISA方法测各因子处理组培养基中上清雄激素含量。最后结合以上数据用统计软件作图分析。结果:在调控ILCs增殖方面,LH可调控ILCs往ALCs增殖分化,因为ILCs向ALCs分化过程中只增殖一次,所以与阴性对照组相比,LH处理组中细胞数量并没有显著的增加。FGF19可促进ILC增殖,以10 ng/ml FGF19组最显著,约为阴性对照组相的1.19倍。而FGF1、FGF9、FGF10可抑制ILC增殖,具有细胞毒性的作用,且呈剂量依赖性。其中,FGF10处理组最为显著,10 ng/ml和100 ng/ml处理组的细胞数量分别约为对照组的65.07%和32.57%。上述表明FGFs可调控ILCs增殖。在调控ILCs分化方面,(1)FGF1、FGF6、FGF8b能有显著促ILCs合成睾酮,其中FGF1最显著,10 ng/ml和100 ng/ml处理组睾酮量约为阴性对照组的3.42和3.66倍,这些结果表明,FGF1、FGF6、FGF8b具有促ILCs分化的功能。其余因子组均无显著抑制ILCs分化的功能。(2)加入10 ng/ml LH诱导3小时后发现FGF5、FGF7、FGF20、FGF22显著促进ILCs合成睾酮,其中FGF20最显著,10 ng/ml和100 ng/ml处理组睾酮量分别约为阴性对照组的2.49和3.60倍。表明在LH的诱导下FGF20能更好的促ILCs分化。而FGF9在LH的诱导下呈双向调节作用即低浓度时促进ILCs合成睾酮,而高浓度时则展示抑制。上述表明FGFs和LH可共同调控ILCs合成雄激素。在调控ALCs合成雄激素方面,(1)与阴性对照组产生睾酮含量相比,在因子中100 ng/ml FGF1、FGF17、FGF22均促进ALCs合成睾酮,FGF1最显著,为对照组的约1.58倍;FGF5、FGF6、FGF16、FGF20、FGF21均能够抑制ALCs合成雄激素,其中FGF5最显著,10 ng/ml和100 ng/ml组睾酮含量分别为对照组的约26.26%、56.50%;(2)加入100 ng/ml LH诱导3小时后发现,与阴性对照组相比,FGF1、FGF2、FGF4、FGF6、FGF9、FGF16、FGF18、FGF21均可促进ALCs合成雄激素,其中100 ng/ml FGF1最显著,为阴性对照组的睾酮量的约2.94倍。FGF7、FGF17可抑制ALCs合成雄激素,以100 ng/ml FGF17最显著,为对照组的约41.75%。上述表明FGFs和LH可共同调控ALCs的雄激素合成。结论:睾丸间质细胞在分化发育过程中,受到多种FGFs的调控,如FGF19可促进ILCs的增殖,而FGF1和FGF10能导致ILCs数量的下降;FGF1、FGF6和FGF8b可促ILCs分化;FGF1、FGF2、FGF4、FGF6、FGF9、FGF16、FGF18和FGF21可促进ALCs合成雄激素,而FGF7、FGF17可抑制ALCs合成雄激素。FGFs也可以配合LH调控不同发育时期睾丸间质细胞的雄激素合成能力。这些FGFs可能是日后治疗男性性腺功能减退等相关疾病的关键靶标。更多还原

【Abstract】 Objective:Analyze the regulation of fibroblast growth factor on the proliferation,differentiation and androgen synthesis of rat testicular Leydig cells,and provide preliminary treatment basis for understanding the development of the testicular stromal cell lineage and the treatment of hypogonadism and other diseases.Method:Using Percoll density gradient centrifugation,primary ILCs was isolated from SD rats at 35 days.After 4 hours of adherence treatment,the cells were treated with FGFs of 10 ng/ml and 100 ng/ml,respectively in 48 hours.A high-content microscope was used to analyze the proliferation of ILCs and count the cells;the ELISA method was used to measure the androgen content of the supernatant in the culture medium of each factor treatment group.In addition,using the same density gradient method,ALCs was isolated from 90-day-old SD rats,and the cells were treated with FGFs of10 ng/ml and 100 ng/ml for 24 hours.The ELISA method was used to measure the culture of each factor treatment group.Androgen content in the supernatant of the base.Finally,combined the above data with statistical software to graph and analyze.Result:In terms of regulating the proliferation of ILCs,LH can regulate the proliferation and differentiation of ILCs to ALCs,because ILCs only proliferates once during the differentiation process of ALCs.Compared with the negative control group,the number of cells in the LH treatment group did not increase significantly.FGF19 can promote the proliferation of ILCs,the 10 ng/ml FGF19 group is the most significant,which is about 1.19 times that of the negative control group.FGF1,FGF9,and FGF10 can inhibit the proliferation of ILCs and have cytotoxic effects in a dose-dependent manner.Among them,the FGF10 treatment group was the most significant,and the number of cells in the 10 ng/ml and 100 ng/ml treatment groups was approximately65.07% and 32.57% of the control group,respectively.The above indicates that FGFs can regulate the proliferation of ILCs.In terms of regulating ILCs differentiation,(1)FGF1,FGF6,and FGF8 b can significantly promote the synthesis of testosterone by ILCs,among which FGF1 is the most significant.The amount of testosterone in the 10 ng/ml and 100 ng/ml treatment groups is about 3.42 and 3.66 times that of the negative control group.These results It shows that FGF1,FGF6 and FGF8 b have the function of promoting ILCs differentiation.The rest of the factor groups did not significantly inhibit ILCs differentiation.(2)After 3 hours of induction with 10 ng/ml LH,it was found that FGF5,FGF7,FGF20,and FGF22 significantly promote the synthesis of testosterone by ILCs,among which FGF20 was the most significant,and the testosterone levels in the 10 ng/ml and 100 ng/ml treatment groups were about the negative control respectively 2.49 and 3.60 times of the group.It shows that FGF20 can better promote ILCs differentiation under the induction of LH.However,FGF9 exhibits a two-way regulatory effect under the induction of LH,that is,it promotes ILCs to synthesize testosterone at low concentrations,and exhibits inhibition at high concentrations.The above shows that FGFs and LH can jointly regulate the synthesis of androgens by ILCs.Regarding the regulation of ALCs synthesis androgen,(1)Compared with the negative control group,100 ng/ml FGF1,FGF17,and FGF22 promote ALCs to synthesize testosterone.FGF1 is the most significant,which is about 1.58 times that of the control group;FGF5,FGF6,FGF16,FGF20,FGF21 can inhibit the synthesis of androgens by ALCs,among which FGF5 is the most significant,the testosterone content of 10 ng/ml and 100 ng/ml groups is about 26.26% and 56.50% of the control group respectively;(2)adding 100ng/ml LH induction after 3 hours,it was found that compared with the negative control group,FGF1,FGF2,FGF4,FGF6,FGF9,FGF16,FGF18,FGF21 can promote the synthesis of androgens by ALCs,and 100ng/ml FGF1 is the most significant,which is the negative control group.About 2.94 times the amount of testosterone.FGF7 and FGF17 can inhibit the synthesis of androgens by ALCs,with 100 ng/ml FGF17 being the most significant,which is about 41.75%of the control group.The above shows that FGFs and LH can co-regulate androgen synthesis of ALCs.Conclusion:During the differentiation and development of testicular stromal cells,they are regulated by a variety of FGFs.For example,FGF19 can promote the proliferation of ILCs,while FGF1 and FGF10 can lead to a decrease in the number of ILCs;FGF1,FGF6 and FGF8 b can promote ILCs differentiation;FGF1,FGF2,FGF4,FGF6,FGF9,FGF16,FGF18 and FGF21 can promote the synthesis of androgens by ALCs,while FGF7 and FGF17 can inhibit the synthesis of androgens by ALCs.FGFs can also coordinate with LH to regulate the androgen synthesis ability of Leydig cells at different developmental stages.These FGFs may be the key targets for the treatment of male hypogonadism and other related diseases in the future.更多还原