【作者】 邹海军；

【导师】 王杏龙；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 为了研究生精细胞和支持细胞在体外的生长特性、形态特征以及这两种细胞之间的相互关系,本实验以成年公兔的睾丸为实验材料,采用不同的酶处理方法对睾丸精细管进行消化,然后利用低渗液进行处理和选择性贴壁培养,对支持细胞进行纯化,并且还对支持细胞进行培养和鉴定;同时,本实验还通过Percoll密度梯度离心和选择性贴壁培养的方法,对生精细胞进行纯化;最后,将纯化的生精细胞单独培养,同时还将纯化的生精细胞与支持细胞进行共培养。结果如下:1采用两种消化方法,均能得到单细胞悬液,并且单个睾丸所获细胞总数分别为5.68×108个和4.07×108个,二者之间差异不显著(P>0.05);所得细胞悬液中圆形细胞比率分别为86.66%和79.21%,两者之间差异也不显著(P>0.05);就细胞存活率而言,两者差异显著(95.28%VS 82.91%, P<0.05)。2利用20 mM Tris-HCl对细胞悬液进行低渗处理后,支持细胞的比率升高,与处理前相比,差异显著(23.31% VS 16.01%, P<0.05);但细胞的存活率降低,与处理前相比差异极显著(80.98% VS 95.96%,P<0.01)。3将低渗处理后的支持细胞悬液和未经处理的细胞悬液都进行选择性贴壁培养,结果表明,两种细胞悬液中的支持细胞比率都显著升高。经低渗处理的细胞悬液培养后与培养前相比,支持细胞的比率二者间差异极显著(76.01% VS 23.31%, P<0.01),细胞存活率二者间差异显著(88.02% VS 80.89%, P<0.05);未经处理的细胞悬液培养后,支持细胞的比率也升高了,与培养前相比差异显著(42.86% VS 16.01%,P<0.05),而细胞存活率有所下降,但差异不显著(90.48% VS 95.96%,P>0.05)。此外,将这两种细胞悬液培养后的支持细胞比率进行比较发现,二者间差异极显著(76.01% VS 42.86%,P<0.01)。4消化得到的细胞悬液经Percoll密度梯度离心后,检测发现,生精细胞主要在25%—35%梯度带,其中主要含有生精细胞和支持细胞,它们的比率分别为78.63%和13.11%,此细胞带中细胞的存活率为97.46%。将25%~35%梯度带细胞经选择性贴壁培养后,生精细胞比率达到94.37%,其中还含有5.13%未成熟的精细胞。5纯化的生精细胞单独培养,细胞不能贴壁和增殖,培养4天后,几乎全部死亡;而与支持细胞共培养2天后,细胞开始增殖,7天后达到增殖高峰,两周后,细胞死亡。更多还原

【Abstract】 In order to study the growth characteristic、cell morphologic and the relationship of spermatogenic cells and Sertoli cells, adult male rabbits` testis were used as material of experiment. The testis tissue was digested with different enzyme combinations, the production which contains spermatogenic cells and sertoli cells were treated with hypotonic shock and selective culture, through those methods, Sertoli cells were purified. Then Sertoli cells were cultured and identified. At the same time, the digesting production was treated with Percoll discontinue density gradient centrifugation and selective culture. Then, spermatogenic cells were purified. At last, the high purity spermatogenic cell was cultured single and with sertoli cell, respectively. The results of experiment were as follows:1 Both of two methods had successfully got single cell suspension. The total cells from single testis were 5.68×108 and 4.07×108, respectively, there was no significant difference between two methods (P>0.05). The round cell rate in the single cell suspension were 86.66 % and 79.21 % respectively, there was also no significant difference between two methods (P>0.05). But the live cell rate showed significant difference (95.28% VS 82.91%, P <0.05).2 The single cell suspension was treated with hypotonic shock, after that, the Sertoli cell rate was higher than before (23.31% VS 16.01%, P<0.05). But the live cell rate was lower than before (80.98% VS 95.96%,P<0.01).3 When cultured the two kinds of cell suspensions which were treated with hypotonic shock and no treated, as a result, the Sertoli cell rates in two kinds of cell suspensions were higher than before, both of which showed significant difference, (76.01% VS 23.31%, P<0.01 ) and (42.86% VS 16.01%,P<0.05), respectively. The live cell rate in the treated cell suspension was higher than before cultured, the difference was significant (88.02% VS 80.89%, P<0.05). In the untreated cell suspension, the live cell rate reduced, but the difference was not significant (90.48% VS 95.96%,P>0.05). Moreover, after cultured, the Sertoli cell rate in two kinds of cell suspensions showed significant difference (76.01% VS 42.86%,P<0.01).4 The single cell suspension was treated with Percoll discontinue density gradient centrifugation, and then, every cell line was checked, but spermatogenic cell almost existed in the cell line which was between the 25 % and 35 % Percoll. In that cell line, spermatogenic cell rate was 78.63 %. Besides spermatogenic cell, there was sertoli cell also, and the rate was 13.11 %. The total live cell rate in that cell line was 97.46 %. After selective cultured, that cell line contained spermatogonic cells and spermatid (94.37 % and 5.13 %, respectively).5 When the purified spermatogenic cells were cultured by single, they could not anchor and reproduce neither. Four days later, almost all of cells were dead. But the spermatogenic cells cultured with Sertoli cells begin reproduced after two days, they reached the highest multiplication at the seventh day. Two weeks later, all of cells were dead.更多还原