分子遺傳方法在偵測癌細胞基因突變上之應用

Abstract

腫瘤是由一個基因變異的細胞不斷增生而造成，其形成的過程為多重步驟。隨著腫瘤的進展，會有更多的基因變異累積而使腫瘤更趨惡化。致癌基因和腫瘤抑制基因的突變與腫瘤形成有極為密切的關係，因此偵測這兩種基因是否發生變異可用來做為癌症在臨床上診斷的指標。根據統計，70％大腸直腸癌病患其腫瘤組織具有變異的P53腫瘤抑制基因，本文即以不同的分子遺傳方法來偵測大腸直腸癌組織中P53基因的突變情形。在此研究模式中，我們以聚合?連鎖反應（PCR）以及南氏轉印法和限制片段長度多形性（RFLP）等方法偵測P53基因是否有缺失和插入的現象。利用反轉錄-聚合?連鎖反應（RT-PCR）來偵測訊息的表現，並且以單股核酸型態多形性（SSCP）檢視基因的點突變。經過PCR和RT-PCR的分析，我們發現第95號檢體腫瘤組織的p53基因無法做有效的增幅，因而懷疑其基因可能有缺失發生。從南氏轉印和RFLP的結果也可以看到其p53基因有不同長度的限制片段。第97號檢體腫瘤組織的p53基因以PCR, RT-PCR和RFLP分析後，與正常組織的p53基因相比雖然看不出任何差異，但是在SSCP的分析中，其p53基因在電泳時移動的位置卻與其他檢體不同。為了證明移動位置的不同是因為點突變而造成，我們以核酸定序分析而確定其p53基因在第248密碼子發生點突變，向代表精胺酸的密碼子CGG突變成代表色胺酸的TGG。由上述結果可知，利用PCR和SSCP來偵測基因突變具有很高的效率和可信度，但是PCR和RFLP不能偵測到基因的點突變，故這些方法必須互相配合使用，才能正確的偵測出基因是否發生變異。 RDA是一種用來分析兩個基因組間之差異的方法，由於腫瘤形成並非單一基因變異即可造成，其包含了許多基因的變異，因此我們希望能利用RDA分析腫瘤組織與正常組織基因組彼此間的差異。本文先以小老鼠肝臟DNA與λ噬菌體DNA之混合做為建立RDA實驗系統的模式，再以小老鼠肝臟DNA和RL♂l細胞株DNA這兩個已知有差異存在的基因組來測試RDA的實驗系統，從而確定本文所建立的系統可以有效的分析兩個基因組之間的不同。我們可以利用RDA研究是否還有其他已知或未知的基因突變發生在腫瘤形成的過程中。未來，RDA也可用來做為研究遺傳疾病之工具。

Tumorigenesis is a multistage process starting from a clonal expansion of the cells that gain a selective growth advantage by genetic (or epigenetic) events in the cells. During progression, further genetic alterations accumulate in the cells. Molecular genetic studies of human cancer cells have carried multiple genetic alterations, including alterations of oncogenes and tumor suppressor genes. The identification and characterization of genes mutation in cancer cell would be a good indicator in diagnosis. It is believed that a tumor suppressor gene, p53, plays a central role in the genesis and progression of human cancers. Researchers have documented about 70% of colorectal cancer carries p53 mutations. Thus, we applied different molecular genetics methods to detect p53 mutations in colorectal cancer cells. In this model, polymerase chain reaction (PCR), southern blot and restriction fragment length polymorphism were used for the detection of DNA deletion or insertion. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of messenge RNA (mRNA), and point mutation was detected by single-strand conformational polymorphism (SSCP). We found the p53 gene in tumor tissue of sample 95 could not be efficiently amplified by PCR, and its patterns of RFLP were different from normal tissue. It was possibly resulting from gene deletion. In SSCP analysis, the p53 gene in tumor tissue of sample 97 shifted specifically on electrophoresis. After sequencing determination, the position of point mutation was in the 248 codon which was changed CGG to TGG. In our results, PCR and SSCP were useful and powerful methods to detect gene mutation. However, point mutation was not detectable by southern blot. Because of accumulation of genes mutation in the process of the tumorigenesis, we present here a general method, representational difference analysis (RDA), for finding differences between normal and tumor genomes. In the first set of stringent tests of our protocols, we added λ phage DNA to mous liver DNA to create a model tester, and used the same DNA without λ phage DNA as driver. Then, we used RL♂1 DNA as tester and mouse liver DNA as driver to test the system which was described above. In the future, RDA could be used to detect gene mutation in human cancer and could be applied in the studies of genetic disorder. 4