口服抑制素基因疫苗对大鼠精子发生的作用研究

发布时间：2018-04-16 15:30

本文选题：抑制素 + 基因免疫　；参考：《湖南农业大学》2016年硕士论文

【摘要】：本研究采用酶联免疫测定、组织切片技术、荧光定量PCR和蛋白免疫印迹等方法与手段,分析口服新型非抗性抑制素基因疫苗对雄性大鼠精子发育、睾丸组织结构、激素水平及精子发生相关基因和蛋白的表达作用,探究新型抑制素基因疫苗对大鼠精子发生的作用机制。1口服抑制素基因疫苗对雄性大鼠免疫应答的影响用不同菌落数新型抑制素基因疫苗PXAIS (1010CFU/mL,109CFU/mL,108CFU/mL)各4mL口服免疫大鼠,初次免疫二周后加强免疫二次,用ELISA检测不同时期血清抗体吸光值。研究结果表明：(1)三个免疫组血清抗体P／N值均有不同程度的升高,中剂量组在第一次加强免疫后第一周P／N值(1.64+0.21)与对照组相比有极显著差异(P0.01);(2)高剂量组在第一次加强免疫后第一周P／N值(1.49+0.30)与对照组有显著差异(P0.05);(3)低剂量组在第一次加强免疫后第一周P／N值(1.56+0.35)与对照组相比有显著差异(P0.05)。2口服抑制素基因疫苗对雄性大鼠精子发育的影响断颈处理大鼠以检测大鼠脏器系数、脏器体积、精子数和畸形率的变化,研究结果表明：(1)实验组睾丸体积明显升高,且低剂量在和中剂量组大鼠睾丸体积与对照组相比差异显著(P0.05),但对对附睾体积没有明显影响(P0.05);(2)高剂量组睾丸系数(0.93±0.06)与对照组相比差异极显著(P0.01),低剂量组附睾系数(0.27±0.02)与对照组相比差异显著(P0.05),各实验组精囊腺脏器系数与对照组相比没有显著影响(P0.05);(3)实验组精子数量升高,畸形率降低,其中高剂量组精子数量(8.41+0.63)和畸形率(19.88+2.72)与空质粒组相比差异显著(P0.05),且中剂量组精子数量(9.58+2.19)和畸形率(18.50+2.30)与空质粒组相比有极显著差异(P0.01)。3口服抑制素基因疫苗对雄性大鼠睾丸组织结构的影响HE染色后在光学显微镜下观察大鼠睾丸组织形态学变化,研究结果表明：(1)各组睾丸组织生精小管外有完整的基膜,生精细胞发育正常,层次较清楚；(2)各组生精小管内生精细胞数量不同,中剂量组和对照组相比,生精细胞层数增加,管壁基膜变厚,官腔空隙变窄,腔隙中胞质残余增加。4口服抑制素基因疫苗对雄性大鼠内分泌激素水平的影响本实验采用ELISA技术检测各组不同时期血清中抑制素B(INHB)、促卵泡素(FSH)、黄体生成素(LH)和睾酮(T)激素水平变化,研究结果表明：(1)免疫后三个实验组血清抑制素B水平均有不同程度降低,低剂量组在初次免疫后第二周(15.66+0.51)与对照组相比差异显著(P0.05),中剂量组在第一次加强免疫后第一周(14.414-1.10)出现最低值,并与对照和空质粒组相比差异极显著(P0.01);(2)免疫后各实验组FSH水平与对照组相比明显升高,初次免疫后第二周,中剂量组(6.02+0.12)和高剂量组(5.92+0.35)与对照组和空质粒组相比差异极显著(P0.01),在免疫后第三周里,低剂量组(5.53+0.16)与对照组相比差异显著(P0.05);(3)与对照组相比,低剂量和中剂量组LH有所降低,初次免疫后第一周中剂量组LH水平(14.48+0.67)与空质粒组相比差异显著(P)0.05),初次免疫后第二周LH含量继续下降,中剂量组(14.01+0.63)和低剂量组(13.84±0.40)与空质粒组差异显著护0.05),低剂量组与对照组差异显著(P0.05);(4)对照组和空质粒组从第一周到第七周睾酮(T)分泌水平变化不明显,低剂量组第一次加强免疫后第二周(74.82±4.50)与对照组差异显著(P0.05),其余周次均无明显变化,初次免疫第一周中剂量组水平(76.41±3.75)升高,且与对照组相比差异显著(P)0.05),中剂量组初次免疫后和第一次加强免疫后第二周(86.57±6.29)与对照组和空质粒组相比均差异极显著(P0.01)。5口服抑制素基因疫苗对雄性大鼠精子发生相关基因和蛋白的影响采用RT-PCR和蛋白免疫印迹分别检测大鼠睾丸组织中INHβB、Vimentin、SMAD4基因在mRNA和蛋白水平的表达变化,研究结果表明：(1)抑制素基因疫苗免疫大鼠能降低INHβB亚基表达,中剂量组(19.40±3.60)和对照组(23.13+1.02)mRNA表达水平差异显著(P0.05)；中剂量组和高剂量组蛋白表达水平与对照组相比均差异极显著(P0.01)；低剂量组INHβB蛋白表达水平与对照组差异显著(P0.05)。(2)各免疫组Vimentin mRNA水平表达升高,三个实验组与对照组相比均有统计学意义,其中低剂量组(20.14+2.74)与对照组(17.65±1.49)差异显著(P0.05),中剂量组(22.01±1.60)与对照组和空质粒组相比均差异极显著(p0.01),高剂量组(21.09士2.11)与对照组和空质粒组相比差异显著(P0.05)；各免疫组Vimentin蛋白水平表达升高,且均与对照组差异极显著(p0.01)；(3)中剂量组和高剂量组Smad4 mRNA表达升高,中剂量组(25.32±3.11)与对照组(19.93±1.15)和空质粒组(19.92±0.89)相比均差异极显著(P0.01),高剂量组(22.36±1.98)与对照组和空质粒组以及中剂量组相比均有显著差异(P0.05),低剂量组与对照组无明显差异((P0.05))；免疫组Smad4蛋白表达水平均上调,中剂量组和高剂量组与对照组相比均有极显著差异(P0.01),低剂量组与对照组差异显著(P0.05)。综上,一方面,抑制素基因疫苗能引起雄性大鼠的免疫应答,升高精子数量,降低精子畸形率,促进精子发育；另一方面,免疫降低了雄性大鼠内分泌激素抑制素B和LH的的分泌,升高FSH和T的分泌,并上调了Smad4、Vimentin基因mRNA和蛋白表达的水平,下调了INHβB亚基基因mRNA和蛋白表达的水平,说明抑制素基因疫苗免疫能通过引起雄性大鼠内分泌激素水平和基因蛋白表达的变化,改善雄性大鼠的精子发生。
[Abstract]:The results showed that : ( 1 ) The serum antibody P / N of the experimental group was significantly higher than that in the control group ( P0.05 ) ; ( 3 ) The number of sperm in the high dose group was higher than that in the control group ( P0.05 ) .
Compared with the control group , the levels of LH , FSH , LH and T were significantly higher than those in the control group ( P < 0.05 ) .
Compared with the control group , the expression level of high - dose group and high - dose group was significantly higher than that in the control group ( P0.01 ) .
( 2 ) The level of vimentin mRNA was significantly higher in the low - dose group ( 20.14 + 2.74 ) than that in the control group ( P < 0.01 ) .
The expression of Vimentin in each group was significantly higher than that in the control group ( p < 0.01 ) .
( 3 ) Compared with the control group ( 19.93 卤 1.15 ) and the empty plasmid group ( 19.92 卤 0.89 ) , the expression of Smad4 mRNA in the middle dose group and the high dose group was significantly higher than that in the control group ( 19.93 卤 1.15 ) and the empty plasmid group ( 19.92 卤 0.89 ) ( P < 0.01 ) .
The expression level of Smad4 protein in the vaccinated group was up - regulated , the middle - dose group and the high - dose group had significant difference compared with the control group ( P0.01 ) , the difference between the low - dose group and the control group was significant ( P0.05 ) . On the one hand , the inhibition of the gene vaccine could induce the immune response of the male rats , increase the sperm count , reduce the sperm deformity rate and promote the development of the sperm ;
On the other hand , immunity decreased the secretion of hormone B and LH in male rats , increased the secretion of FSH and T , up - regulated the level of Smad4 and Vimentin gene mRNA and protein expression , downregulated the level of mRNA and protein expression of INH尾B subunit gene , and indicated that the immunity of the gene vaccine could improve the sperm production of male rats by inducing changes of endocrine hormone level and gene protein expression in male rats .

【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S814.1

【参考文献】