人羊膜间充质干细胞改善高龄小鼠睾丸功能的实验研究

doi:10.12280/gjszjk.20210138

摘要/Abstract

摘要：

目的: 探讨人羊膜间充质干细胞（hAMSC）改善高龄雄性小鼠睾丸功能的作用。方法: 以36周龄雄性小鼠为高龄模型组,10周龄小鼠为年轻对照组。31只高龄模型组小鼠随机分为5组,分别为生理盐水对照组、1%人血清白蛋白对照组和治疗组,其中生理盐水对照组3只,其余各组每组7只。治疗组小鼠尾静脉注射hAMSC,剂量分别为3.4×10⁶细胞/kg（低剂量组）、6.7×10⁶细胞/kg（中剂量组）、1.4×10⁷细胞/kg（高剂量组）。每周注射1次,治疗4次后小鼠继续饲养,5周后取血检测小鼠血清睾酮水平;行附睾内精子的分析;免疫荧光检测睾丸组织内STEM121和CD105的表达和定位,观察睾丸组织病理学改变,检测类固醇激素生成急性调节蛋白（StAR）、17β-羟基类固醇脱氢酶（17β-HSD）的表达水平。结果: 与年轻雄鼠比较,治疗前高龄雄鼠体质量增加、睾丸体质比下降、睾周脂肪质量增加（均P<0.01）,血清睾酮水平下降（P<0.05）,精子活动率降低、正常形态精子率下降（均P<0.05）。治疗前高龄雄鼠的精子计数、睾丸组织StAR和17β-HSD蛋白表达水平与年轻雄鼠比较差异无统计学意义（均P>0.05）。hAMSC治疗后,睾丸间质区可见STEM121和CD105表达的细胞;高剂量组血清睾酮水平升高（P<0.05）;睾丸组织StAR蛋白表达无显著差异（P>0.05）,但高剂量组17β-HSD蛋白表达上调（P<0.05）。中剂量组和高剂量组睾丸组织的生精小管细胞层数较人血清白蛋白对照组增多（P<0.05）。结论: 初步实验结果表明,hAMSC对睾丸衰老具有保护作用,并促进雄激素合成。

关键词: 间充质基质细胞, 羊膜, 衰老, 生育力, 间质干细胞移植, 人羊膜间充质干细胞, 睾丸衰老, 生精功能, 睾酮合成

Abstract:

Objective: To explore whether human amnion mesenchymal stem cells(hAMSC) can improve testicular steroidogenesis in the aging mice. Methods: 31 male mice aged 36-weeks as the model of aging males were randomly divided into 5 groups, with 3 mice in the normal saline control group and 7 mice in the other four groups. Different doses of hAMSC (3.4×10⁶ cells/kg, 6.7×10⁶ cells/kg, 1.4×10⁷ cells/kg) were injected through the tail vein, and the 1% human serum albumin group was used as the treatment control. And the normal saline was used as the blank control. Seven mice aged 10 weeks were used as the young control. The mice were injected four times and once a week. After five weeks of recovery, both testes were taken. The histopathological change was observed by HE staining. The expressions of StAR and 17β-HSD in testicular tissues were detected by immunoblotting. The expressions of STEM121 and CD105 were also detected by immunofluorescence. The epididymis was taken to evaluate sperm count, sperm motility rate and sperm morphology. The level of testosterone was tested by chemiluminescence immune assay.Results: Compared with the young control, the aged mice in the blank control and the treatment control had the increased body weight, the decreased testicular mass-to-weight ratio, the increased fat around the testes (all P<0.01), and the decreased sperm motility rate and normal morphological sperm rate (bothP<0.05). There were no significant differences in sperm density and the expressions of StAR and 17β-HSD protein in testicular tissues between the aging control and the young control (allP>0.05). After five weeks of the hAMSC treatment, the immunofluorescence images showed that there were many cells with the positive expressions of STEM121 and CD105 in the interstitial area of testis. There were no significant differences in the expression of StAR in testis among the treatment groups. The expression of 17β-HSD in the high-dose group was increased than that in the treatment-control group, and the level of testosterone was also increased (bothP<0.05). The histopathology showed that the cell layers of seminiferous tubules were increased in the medium dose group and the high dose group than those in the treatment control group (P<0.05).Conclusions: The preliminary results indicate that hAMSC has a protective effect on testicular aging and can improve testicular steroidogenesis in the aging mice.

Key words: Mesenchymal stromal cells, Amnion, Aging, Fertility, Mesenchymal stem cell transplantation, Human amnion mesenchymal stem cells, Testicular aging, Spermatogenic function, Steroidogenesis

张月鑫, 刘菡文, 施陈楠, 宁松, 周静, 李楚玉, 杨晓玉, 覃莲菊, 刘嘉茵, 胡艳秋, 崔毓桂. 人羊膜间充质干细胞改善高龄小鼠睾丸功能的实验研究[J]. 国际生殖健康/计划生育, 2021, 40(4): 265-271.

ZHANG Yue-xin, LIU Han-wen, SHI Chen-nan, NING Song, ZHOU Jing, LI Chu-yu, YANG Xiao-yu, QIN Lian-ju, LIU Jia-yin, HU Yan-qiu, CUI Yu-gui. Human Amnion Mesenchymal Stem Cells Improve Testicular Steroidogenesis in Aging Mice[J]. Journal of International Reproductive Health/Family Planning, 2021, 40(4): 265-271.

图/表 11

参考文献 21

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组别	n	体质量（g）	睾丸体质比	睾周脂肪质量（g）	血清睾酮（nmol/L）	StAR/ β-actin	17β-HSD/ GAPDH	精子计数（个/mL,×10⁶）	精子活动率（%）	正常形态精子率（%）	生精小管直径（μm）	生精小管细胞层数
年轻对照组	7	24.33±0.33	0.74±0.02	0.33±0.00	46.44±9.05	0.61±0.24	0.49±0.08	5.80±0.59	72.93±8.07	95.00±1.73	146.90±55.48	8.20±1.40
治疗前高龄组	7	37.67±0.88	0.57±0.02	2.42±0.27	12.91±2.25	0.65±0.31	0.44±0.18	5.88±1.58	52.57±9.25	85.43±2.76	190.90±27.07	5.80±1.22
t		14.14	4.86	7.61	3.60	0.23	0.51	0.08	2.87	5.09	3.92	7.09
P		0.00	0.01	0.00	0.01	0.82	0.63	0.94	0.04	0.01	0.00	0.00

组别	n	体质量（g）	睾丸体质比	睾周脂肪质量（g）	血清睾酮（nmol/L）	StAR/ β-actin	17β-HSD/ GAPDH	精子计数（个/mL,×10⁶）	精子活动率（%）	正常形态精子率（%）	生精小管直径（μm）	生精小管细胞层数
年轻对照组	7	24.33±0.33	0.74±0.02	0.33±0.00	46.44±9.05	0.61±0.24	0.49±0.08	5.80±0.59	72.93±8.07	95.00±1.73	146.90±55.48	8.20±1.40
治疗前高龄组	7	37.67±0.88	0.57±0.02	2.42±0.27	12.91±2.25	0.65±0.31	0.44±0.18	5.88±1.58	52.57±9.25	85.43±2.76	190.90±27.07	5.80±1.22
t		14.14	4.86	7.61	3.60	0.23	0.51	0.08	2.87	5.09	3.92	7.09
P		0.00	0.01	0.00	0.01	0.82	0.63	0.94	0.04	0.01	0.00	0.00

组别	n	血清睾酮	StAR/β-actin	17β-HSD/GAPDH
年轻对照组	7	46.44±9.05	0.61±0.10	0.49±0.04
治疗前高龄组	7	12.91±2.25	0.65±0.13	0.44±0.09
生理盐水对照组	3	16.87±3.31*	0.70±0.21	0.51±0.11
F		9.79	0.13	0.27
P		0.01	0.88	0.77

组别	n	血清睾酮	StAR/β-actin	17β-HSD/GAPDH
年轻对照组	7	46.44±9.05	0.61±0.10	0.49±0.04
治疗前高龄组	7	12.91±2.25	0.65±0.13	0.44±0.09
生理盐水对照组	3	16.87±3.31*	0.70±0.21	0.51±0.11
F		9.79	0.13	0.27
P		0.01	0.88	0.77

组别	n	血清睾酮	StAR/β-actin	17β-HSD/GAPDH
生理盐水对照组	3	16.87±3.31	0.70±0.21	0.51±0.11
人血清白蛋白对照组	7	14.85±2.72	0.54±0.18	0.49±0.11
低剂量组	7	18.36±3.41	0.49±0.19	0.48±0.19
中剂量组	7	15.24±1.92	0.55±0.17	0.71±0.10
高剂量组	7	28.51±9.40**	0.84±0.33	0.82±0.09*
F		5.56	1.63	4.53
P		0.01	0.40	0.02