抑制性消减杂交法筛选植入人发角蛋白与单纯脊髓损伤大鼠基因表达的差异

Difference of gene expression between rats with simple spinal cord injury and those undergoing human hair keratin transplantation by using suppression subtractive hybridization

目的:采用抑制性消减杂交筛选植入人发角蛋白大鼠损伤脊髓基因性差异表达。方法:实验于2004-11在汕头大学中心实验室进行。分别从植入人发角蛋白损伤组与单一损伤对照组4thw组织中提取mRNA,反转录成双链cDNA,经RsaI酶切、接头连接、两次消减杂交和两次抑制性聚合酶链扩增,而后将消减cDNA产物进行T/A克隆和测序分析。结果:所获植入人发角蛋白脊髓损伤组织差异表达基因的消减cDNA产物散布在280～800bp范围。经蓝白筛选和酶切鉴定,共得到232个阳性克隆。对其中20个做测序分析获得了氨基酸转运蛋白系统A2(Ata2)、细胞色素氧化酶Ⅲ(COXⅢ)、抗过氧化物蛋白5(Prd5)、Tap1蛋白和半胱氨酸蛋白酶抑制剂(Cst3)等5个表达蛋白。结论:prd5、Ata2和COXⅢ分别通过参与脊髓损伤后抗氧化损伤过程,起到抑制自由基、兴奋性氨基酸的产生,减轻Ca离子紊乱的程度,降低了继发性损害对脊髓组织的二次损伤程度;而Tap1蛋白和Cst3则参与人发角蛋白的降解过程。

AIM： To clone genes differentially expressed in rats after transplanted human hair keratin to area of spinal cord injury by means of the suppression subtractive hybridization （SSH）. METHODS： The experiment was conducted in the Central Lab of Shantou University during November 2004. The mRNA was isolated from 4%, tissue after transplanted human hair keratin to area of acute spinal cord injury group and single injured control group. And that was just only acute spinal cord injury for reverse transcription to ds cDNA. After Rsal enzyme digestion, adaptor ligation, two rounds of hybridization, and two rounds of suppression polymerase chain reaction （PCR）, then the final subtracted cDNA products were subcloned by T/A and sequenced. RESULTS： The subtracted cDNAs of differentially expressed genes in adult rat after transplanted human hair keratin to area of acute spinal cord injury look like smears from 280 bp to 800 bp. 232 positive clones were obtained from subtracted eDNA library through white-blue visual analysis and enzyme digestion. By sequencing analysis of 20 clones, it was found that five eDNA sequences were special novel genes of amino acid transporter system A2 （Ata2）, cytohromc oxidasc Ⅲ（COXⅢ）, pcroxircdoxins 5 （Prd5）, Tapl protein and cysteine proteinase inbibitor cystatin c （Cst3）. CONCLUSION： Prd 5, Ata2 and COX m played a role on reducing radicle, irritability amino acid, controlling calcium turbulence, and reducing other succeed damage to lighten the damage degree of spinal cord. Tapl protein and Cst3 participated in dispelling human hair keratin.