| Objective:To determine the effect of cholesterol on the separation of testosterone in rat Leydig cells,and to prevent and delay the occurrence of testosterone deficiency diseases.Methods:1)Part Ⅰ: Randomly selected 4 weeks old healthy young male SD 12 rats as the parent.The Leydig cells were isolated from the testis of young SD rats with low concentration of II and type IV collagenase digestion,Purified,cultured and identified.2)Part II: The primary cultured LCs were randomly divided into control group A and experimental group B.Control group A was divided into control group A1 and A2,experimental group B was divided into experimental group B1,B2,B3 and B4.(Control group A2)was incubated with standard medium 2(DMEM / F12 medium,fetal bovine serum 5%,secondary antibody 100,000 U / L,LDL-C 1.0ug / ml)Ml).In the experimental group B1,low density lipoprotein cholesterol(LDL-C)0.2,0.5,1.05 ug was added in the control group A1,respectively.In the experimental group B2,on the basis of the control group A2(ox-LDL-C)0.2,0.5,1.0,5ug were added to the control group A1 on the basis of the control group A1,respectively.The experimental group B3 was treated with LDL-C 0.2,0.5,1.0,5ug;ox-LDL-C 0.2,0.5,1.0 and 5ug were added to the control group A2,respectively.4)After the culture of 24 h,the culture medium was collected and the testosterone was measured by enzyme-linked immunosorbent assay(ELISA).Results: PartⅠ:Identification and purification of Leydig cells by way of low concentration of type II,IV collagenase digestion.The best time for enzyme digestion of Leydig cell with 0.1% type Ⅰand IV collagenase was 1h.The best adherent time for the purification of Leydig cells was 1-2h.After 24 hours of culture,the purity of Leydig cells was about 98.1±1.5%,which was confirmed by immunofluorescence staining with 3β-HSD and the survival rate of Leydig cells was about 97.3±1.6% conformed by trypan blue staining.Adherent cells were cultured for 12 hours,and the synthesis of testosterone reached the peak.PartⅡ:Effects of different concentrations of cholesterol on testosterone secretion in rat Leydig cells cultured in vitro1)In the experimental group B1,the concentration of testosterone was the highest in the medium supplemented with 0.5ug LDL-C compared with 0.2ug,1.0ug and 5.0ug LDL-C group,P <0.05,the difference was statistically Learning meaning.Compared with the control group A1,the concentration of testosterone in the medium supplemented with 0.5 μg LDL-C was higher,P <0.05,the difference was statistically significant.The concentration of testosterone in 0.2 μg and 1.0 μg LDL-C medium was significantly lower than that in control group P> 0.05.There was no significant difference between the two groups.The concentration of testosterone in the medium supplemented with 5ug LDL-C was lower than that in the control group A1,P <0.05,the difference was statistically significant.2)In the experimental group B2,the concentration of testosterone was 0.5 ug and 1.0 μg LDL-C in the culture medium.The concentration of testosterone was the highest,P> 0.05,the difference was not statistically significant,The concentration of testosterone in ug and 5.0 ug LDL-C medium was higher than that in 0.5ug and 1.0ug groups,and the testosterone concentration was higher,P <0.05.The difference was statistically significant.Compared with control group A2,the concentration of testosterone in 0.2ug,0.5ug and 1.0ug LDL-C medium was significantly higher than that in control group,P <0.05.The difference was statistically significant.Compared with control group A2,the concentration of testosterone in 5.0 ug LDL-C medium was significantly decreased,P <0.05,the difference was statistically significant.3)In the experimental group B3: Compared with the control group A1,the concentration of testosterone in the medium supplemented with 0.2 ug and 0.5 ug ox-LDL-C was lower than that of the control group A1,P <0.05,the difference was statistically significant,1.0ug And 5.0 μg group testosterone concentration below 0.15 ng / ml,lower than the standard curve measurement range,the measured value may be a large error,so do not compare.4)In the experimental group B4,the testosterone concentration in the medium supplemented with 0.2ug,0.5ug,1.0ug and 5ug ox-LDL-C was lower than that of the control group A2,P <0.05,the difference was statistically significant,The concentration of testosterone decreased significantly with the increase of ox-LDL-C.5)Compared with experimental group B2,the experimental group B1 showed significant increase in testosterone by LDL-C in experimental group B2,0.5 μg group was added in experimental group B1,testosterone concentration was the highest,ex perimental group B2,0.5 ug and 1.0 ug group testosterone concentration The highest,showing that experimental group B2 can use higher concentrations of LDL-C to synthesize cholesterol.Conclusion:Through high concentration Ⅱ,type Ⅳ collagenase digestion method can get enough high activity of high purity testicular interstitial cells,and LCs proliferation ability.Low concentration of LDL-C can promote the synthesis of testosterone in vitro by LCs.High concentration of LDL-C can inhibit the synthesis of testosterone in LCs in vitro.HCG can enhance the ability of LCs in vitro to synthesize testosterone and increase the concentration of LCs LDL-C was used.The low concentration of ox-LDL-C inhibited the synthesis of testosterone in vitro,and its inhibitory ability increased with the increase of concentration... |
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