目的 建立基于96孔板的Bhas 42細胞轉(zhuǎn)化試驗高通量檢測方法，并探討此方法用于藥物非臨床階段體外致癌性評價的前景。方法 建立Bhas 42細胞轉(zhuǎn)化試驗96孔板高通量檢測方法，使用已知陽性誘癌物苯并芘（Benzoapyrene，BaP）和環(huán)磷酰胺（Cyclophosphamide，CP）為對照品，比較轉(zhuǎn)化灶人工計數(shù)法和過氧化氫高通量96孔板法對結(jié)果判定的影響。進行試驗的細胞在接種后第19天以一定濃度的過氧化氫處理細胞24 h。24 h后，洗去含過氧化氫的培養(yǎng)基，加入新鮮培養(yǎng)基并加入CCK-8染色孵育2~4 h后測定450 nm波長吸光度以確定細胞轉(zhuǎn)化率。同時，轉(zhuǎn)化灶人工計數(shù)法細胞在第21天經(jīng)甲醇固定，吉姆薩染液染色后計數(shù)每孔克隆數(shù)，對比兩種方法的差異性。結(jié)果 轉(zhuǎn)化灶人工計數(shù)法中，與陰性對照組比較，BaP和CP在啟動試驗中均被評價為陽性；過氧化氫高通量法得到與轉(zhuǎn)化灶人工計數(shù)法相同的結(jié)果。結(jié)論 成功建立了基于96孔板的Bhas 42細胞轉(zhuǎn)化試驗的過氧化氫高通量檢測方法，該方法相比于傳統(tǒng)方法可簡單、快速地檢測化合物誘導Bhas42細胞的轉(zhuǎn)化效率，并提高了結(jié)果評判的客觀性。

Objective To establish a high throughput assay for Bhas 42 cell transformation assay based on 96-well plate, and to explore the prospect of this assay in evaluating the carcinogenicity of drugs in non-clinical phase in vitro.Methods A 96-well plate high-throughput assay for Bhas 42 cell transformation assay was established. Benzoapyrene (BaP) and Cyclophosphamide (CP) were used as positive control substances to compare the effects of the manual counting method and 96-well plate high-throughput assay on the results. The Bhas 42 cells were treated with hydrogen peroxide on nineteenth days after inoculation for 24 h. After 24 hours, the medium containing hydrogen peroxide was washed off, fresh medium with CCK-8 was added and incubate for 2-4 hours. The cell transformation rate was determined by measuring 450 nm wavelength absorbance. At the same time, the cells were fixed by methanol on the 21st day and stained by Giemsa solution. The correlation between the two methods was statistically analyzed and compared.Results Compared with the negative control, both BaP and CP were positive in the initiation assay, and the results of high-throughput cell transformation assay were the same as manual counting cell transformation assay.Conclusion A 96-well plate-based high-throughput method for the detection of Bhas 42 cell transformation was successfully established. Compared with the traditional method, this method is easily and quickly, and improves the objectivity of the result evaluation.

上海市科學技術(shù)委員會科研計劃項目（15140901100）