以熱休克蛋白質作為毒性指標可行性之探討

Abstract

當暴露於較正常生長溫度稍高的環境下時，生物體內便會自行誘導產生一種壓力蛋白質：熱休克蛋白質 (heat shock protein, hsp)，以保護其在此溫度下仍能繼續生存，甚至對繼之而來的更高溫度，其存活力亦會提高，此項特性從細菌、動、植物、無脊椎動物甚至於人類皆具備，且為一具高度保存性的遺傳系統。若引申至毒性物質存在的環境下，亦會有類似的情況發生，其所具有的高度保存性顯示此系統對生物體長期以來在有害環境下的成功生存是十分重要的，而其具有的普遍性更透露了以之當作生物指標之可行性。 針對此項特性，本研究計畫以細菌性生物中極為普遍且具代表性之菌種Escherichia coli做為測試物種，選擇重金屬、有機毒物及農藥三種類型共八種毒性物質，針對Heat shock proteins的誘導情形加以觀察，並針對其敏感性與可行性進行一系列之分析及探討。 本研究選用了兩種蛋白質分析方法：Western Blotting及ELISA來分析各種毒物在E. coli體內誘導蛋白質的情形。研究結果顯示，本研究選用之E. coli菌株對這八種不同類型的毒物皆可誘導產生同一種Heat shock protein：hsp70（又名DnaK），而其誘導的趨勢亦大致相同；若將本研究獲得之誘導濃度，與傳統毒性試驗方法比較，皆可發現其誘導濃度明顯低了甚多 (＜10 ppb)；比較以濁度法、平板法及ELISA三種方法求得之NOEC值發現，對重金屬而言，以ELISA可得到較低的NOEC值，即ELISA為一較敏感的方法。若進一步以統計參數F比值及中斷值之計算比較Western Blotting及ELISA兩種方法，結果顯示以ELISA所得數據之組內變異較小，為較精確的實驗方法。故以ELISA來分析E. coli體內hsp70的誘導情形應可做為一敏感且具普遍性之參考依據。

Exposure of cells and organisms to elevated temperatures trigger the synthesis of a small group of proteins called the heat shock proteins (hsps). Their increased expression at moderately high temperatures allows growth and metabolism to continue at the upper end of the cells’ normal temperature range. In most cases increased expression of hsps is also accompanied by increased tolerance to more extreme temperature. It has been observed in a wide variety of bacteria, animals, plants, invertebrate and even to human, and is a highly conserved genetic system. In addition to heat, a number of other stress conditions induced similar or identical reactions. The high evolutionary conservation of this reaction suggests its importance for the survival of cells and tissues under hostile environment conditions. And the generality it has also reveal the practicability of using it as biomarker. According to this property, we plan to use E. coli as test organism for it’s generality and classic, and we select eight chemicals belong to heavy metal, organic toxicant and pesticide, observe the induced condition of heat shock proteins. And then analyze and explore the sensibility and practicability of this method. In this study we choose western blotting and ELISA for protein analysis. Our results show that the different eight toxicants could induce the same kind of heat shock protein: hsp70 (also called DnaK ). They have almost the same induce tendency. Compare the induced concentrations with traditional methods, we find that the induced concentrations we used were very low (＜10 ppb). For the NOEC value of liquid, plate count and ELISA, we can know that using ELISA can gain a smallest NOEC value for heavy metals, means that ELISA is a method with high sensitivity. Further, we calculate statistics parameter F-ratio and cut-off-value for western blotting and ELISA, the results show that ELISA has a smaller within-group variability, means that ELISA is a method with better accuracy. Thus we can say that using ELISA to analysis hsp70 in E. coli could be a method with nice sensitivity and generality.