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Objective: Many studies have shown that the differentiation of the male reproductive system and developmental abnormalities increased significantly, most testicular development, down insufficiency related gubernaculum testis and testicular development, a decrease of close relationship. Human insulin-like factor 3 (INSL3) gene is widely present, the expression of the polypeptide is an insulin-like part of the family of hormones, testicular transabdominal descent stage regulating hormones play a decisive role in the development of the gubernaculum testis. But so far, not been studied. INSL3 impact gubernaculum testis development mechanism The experimental mice gubernaculum testis cells to be cultured in vitro using different concentrations of INSL3 acts directly on cultured mouse gubernaculum testis cells, research INSL3 with cell proliferation and contractile activity of cultured mouse testis cited to explore INSL3 impact testicular descent may Mechanism. Methods: 3d male mice were sacrificed by cervical disinfected after the middle of the cut in the lower abdomen, once again identify male and female, 3 times surgery under a magnifying glass on the bladder both sides complete free the testis, epididymis and gubernaculum testis in PBS tick out most of the gubernaculum testis tissue. The the DMEM culture in of gubernaculum organizations in the containing type I collagenase (1mg/ml) at 37 ° C for sustained concussion digestion 1 hour, 3-4 times the culture medium containing no estrogen serum terminate digestion 1500r / min centrifugal filtrate 5min, the supernatant was removed. The cell pellet without estrogen-containing fetal calf serum (10% v / v) DMEM medium, seeded in 25cm 2 culture flasks, placed in a 5% CO 2 37 ° C saturated humidity incubator static culture. Depending on the testing requirements, the adherent testicular cited with cells 6 × 10 4 / ml density of cell suspension (cells I or II cells) were passaged was inoculated into 6-well plates, 96-well plate and the flask, and by trypan blue staining counting method to determine cell viability, the serum concentrations of 5% of the culture. Approximately 24 hours after the cells were seeded, were randomly divided into experimental group (INSL3 0.02ng/ml, 0.20ng/ml, 2.00ng/ml, 20.00ng/ml), normal control group cells. After dosing, INSL3 for different time specimens were used HE staining, CCK-8, immunofluorescence and flow cytometry analysis technique to detect mouse testis cited with cell proliferation, and F-actin expression. Results: 1. Primary cultured mouse testicular gubernaculum cells most of fibroblast-like cells and a few epithelioid cell growth, radial, flame-like or whorled running cell body fusiform, irregular-shaped or triangular protruding cytoplasmic there. After passage, the cells continued to show into fibroblast-type growth, good homology survival rate is about 90%. 2. CCK-8 found normal control cells showed persistent proliferation, proliferation is increasingly evident after 24h in culture. Similar proliferation trends with the normal control group, the experimental group after dosing 12h exists proliferation (P lt; 0.05), 12h and 24h compared difference is not obvious (P gt; 0.05), 48h significant differences (P lt; 0.05) . Compared with the normal control group, the different dose groups at 12h was no significant difference (P gt; 0.05), 24h and 48h situation is different: 24h when 2.00ng/ml, 20.00ng/ml group compared with the control group were differences (P lt; 0.05), when 0.20ng/ml 48h 2.00ng/ml, 20.00ng/ml group compared with the normal control group were significant differences in the rest of the dose groups is not obvious (P gt; 0.05). 3 immunofluorescence normal control group of F-actin is mainly distributed in the cells surrounding the formation of peripheral actin Ribbon cytoplasm of a small amount of short, thin stress fibers. INSL3 stimulation cytoskeleton remodeling, the cell periphery actin ribbons increased, and microfilament F-actin in the cytoplasm significantly bulky, variable-length, mostly long coarse stress wire, arranged along the cell longitudinal axis more. 20.00ng/ml group change is particularly evident in a dose and time-dependent. Flow cytometry F-actin protein INSL3 role the 24h after 0.02ng/ml, 0.20ng/ml mean fluorescence intensity and the normal control group compared to the decline were statistical differences (P lt; 0.05) , 2.00ng/ml group compared with the control group, no significant differences (P gt; 0.05), 20.00ng/ml mean fluorescence intensity was significantly higher than the control group (P lt; 0.05); 48h after 20.00ng/ml The group mean fluorescence intensity was significantly higher than the normal control group (P lt; 0.05), no significant difference in the remaining dose group compared with the control group. The same dose group, 48h and 24h downward trend which 0.02ng/ml groups had no statistical difference (P gt; 0.05), while 0.20ng/ml 2.00ng/ml, 20.00ng/ml groups were different ( P lt; 0.05). Conclusion: 1. Cultured mouse gubernaculum testis cells were fibroblast-type growth, passaged cell homology, the survival rate is high. 2. CCK-8 test results show gubernaculum INSL3 mouse testis cell proliferation significant role in promoting the dose - time effect. F-actin test results from the morphology, and protein quantitative analysis are prompted INSL3 may be enhanced mouse testis cells shrink to influence the development of testicular gubernaculum gubernaculum, thereby affecting the normal decline in testis.
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