熱休克蛋白質 (Heat-Shock Proteins; HSPs) 或保護子 (Chaperones) 具有保護並穩定在細胞內正常蛋白質的摺疊作用和基本功能。而保護子通常是由很多序列相似性高的成員所組成的大蛋白質家族；但是，成員之間在細胞內的分佈和在基因調控上可能不盡相同。為了區分出保護子的家族各成員蛋白質，我們利用半定量分析的反轉錄脢－聚合脢鏈反應 (RT-PCR) 方法，去分析出人類非小型性肺癌細胞株 (H460 cells) 處在氯化鎘的逆境環境下，所表現出不同熱休克蛋白質七十家族成員基因 (hsp70-1, hsp70-2, hsp70-hom, hsp70-3, grp78, hsp70B, and hsp70B’) 的表現情形，利用一組共同的引子 (primers) 去擴大後，再以不同的專一限制內切脢去區分。已知當細胞受到重金屬鎘的刺激下，細胞的生理反應和基因的表現會因不同濃度和處理時間的長短而有所差異。同理，在我們研究系統中，細胞處於低濃度 (5 uM) 和高濃度 (40 uM to 60 uM) 的鎘刺激下，產生的保護子所表現出的變化情形亦有所不同。我們以硫-35標定分析受刺激的細胞內所新合成的熱休克蛋白質的變化情形，以西方點墨法 (Western blotting) 分析受刺激的細胞內所存在的總熱休克蛋白質七十家族成員的變化情形；再以RT-PCR去分析細胞受到鎘刺激下，個別保護子成員的基因表現。從我們的研究結果指出，這種新的方法確實可以進一步的分析，細胞受到逆境的刺激下，在熱休克蛋白質七十家族成員間，在基因層次的不同變化情形。在此，細胞受到氯化鎘的刺激之下，已知主要的家族成員為hsp70-1、hsp70-2和grp78。除了這幾個成員之外，預期的結果應該可以再分析到另外四個成員(hsp70-hom、hsp70-3、hsp70B和hsp70B’)，而在此狀況下，確實沒有參與在氯化鎘刺激的反應中。

The heat-shock proteins (HSPs) or chaperones can protect and stabilize the folding and functions of normal proteins in cells. Chaperones are generally composed of large, evolutionarily conserved families within which the members are highly related, yet might differ in subcellular localization or gene regulation. We used semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression profile of HSP70 isoforms in human non-small cell lung cancer H460 cells under CdCl2 stress. The expressions of hsp70-1, hsp70-2, hsp70-hom, hsp70-3, grp78, hsp70B, and hsp70B’ were monitored by amplifying from a conserved, common primer set and then differentiated by isoform-specific restriction enzymes. It is known that CdCl2 could elicit different cellular responses and gene expressions in a concentration-dependent and treatment time-dependent manner. Consistent with which, in our system, the expression level or pattern of chaperones varied low concentration (5 uM) and high concentration (40 uM to 60 uM) of CdCl2. We compared the changes of the newly synthesized chaperones by metabolic l ling with [35S]methionine and of the total chaperones by Western blotting analysis in CdCl2 treatment. We further analyzed changes of gene expression of individual isoforms by RT-PCR. From our study, this novel method can be performed to differentiate the members of HSP70 family under stress. Here, it has been found that the main members of HSP70 family in CdCl2-treated cells include hsp70-1, hsp70-2, and grp78. Furthermore, other four members of HSP70 family (hsp70-hom, hsp70-3, hsp70B, and hsp70B’) can be analyzed by this novel method. These four members don’t take part in stress conditions with CdCl2-treated.
二、英文附錄

1. ABSTRACT....................................2

2. INTRODUCTION................................3

3. EXPERIMENTAL PROCEDURES.....................7

4. RESULTS....................................11

5. DISCUSSION.................................18

6. REFERENCES.................................23

7. FIGURE LEGENDS.............................30

8. FIGURES....................................34

9. TABLES.....................................45

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