摘要
目的 筛选鉴定不稳定性心绞痛淋巴细胞差异表达基因 ,从分子水平探讨不稳定性心绞痛的发病机理。为不稳定性心绞痛早期诊断和潜在可能的基因治疗提供线索。方法 应用抑制性消减杂交技术筛选不稳定性心绞痛淋巴细胞和稳定性心绞痛淋巴细胞差异表达基因。将获得的顺向或逆向消减产物与 pGEM TEasyVector连接并转化大肠杆菌JM10 9,采用蓝白斑筛选和菌落PCR筛选有插入片段的克隆。将获得的菌落PCR产物分别等量点在 2张Hybond N +膜的同一位置上 ,同时与顺向消减和逆向消减产物进行斑点杂交 ,进一步筛选差异表达的cDNA片段。以获得的序列表达标签 (EST)为探针 ,与顺向消减和逆向消减产物进行反向Northern杂交 ,将获得的阳性EST测序 ,并进行同源性比较。结果 斑点杂交结果显示 :在不稳定性心绞痛组获得 93个阳性克隆 ,在稳定性心绞痛组获得 112个阳性克隆 ;反向Northern杂交结果显示 :在不稳定性心绞痛组获得 12个阳性EST ,在稳定性心绞痛组获得 2 0个阳性EST ,在各组中随机选取 5个阳性EST测序并进行同源性比较 ,全部为已知基因的部分序列。
Objective To screen and identify the differential expressed genes in lymphocytes from patients with unstable angina, in order to explore the developing mechanism of unstable angina at the molecular level, and to get some clues for diagnoses and potential gene treatment. Methods Suppression subtractive hybridizations (SSH) were performed to screen the differentially expressed genes in lymphocyte RNA between the patients with unstable angina and stable angina. The obtained forward and reverse cDNA fragments were directly inserted into pGEM T Easy vector and transformed into E.coli 109; clones with inserted fragment were screened with blue and white screening and colony PCR. The obtained colony PCR products were spotted at the same position on the Hybond N+ membrane and performed dot blot hybridized with forward and reverse suppression products to further screen the differentially expressed genes. The obtained Expressed Sequence Tags (ESTs) were used as probes to perform Reverse Northern blot with forward and reverse suppression products. The obtained positive ESTs were sequenced and analyzed using BLAST (nr) at NCBI. Results The result of dot blot hybridization showed that 93 positive clones were obtained in unstable angina group; 112 positive clones were obtained in stable angina group. Reverse Northern blot hybridization showed that 12 positive ESTs were obtained in unstable angina group; 20 positive ESTs were obtained in stable angina group.5 positive ESTs in each group were randomly selected to be sequenced and analyzed using BLAST (nr) at NCBI, all of them are sequences of known genes. Conclusion All these ESTs were associated with the occurring and development of unstable angina.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2004年第1期36-39,共4页
Chinese Journal of Cardiology
