栉孔扇贝chlamys farreridna断裂与海水高渗透压关系的探究-study on the relationship between chla mys farreridna fracture and seawater osmotic pressure.docx

3. TUNEL 实验用 TUNEL 法对原代栉孔扇贝血淋巴细胞进行了体外实验，发现：在 465-495nm 波长下，高渗透压下培养的细胞有很强绿色荧光,DNA 断裂情况严重。低渗透压 下培养细胞几乎看不到绿色荧光，DNA 断裂不明显；交换渗透压后，二者在 465-495nm 波长下的 DNA 绿色荧光情况发生互换，表明其 DNA 断裂情况发生了交 换。因此，TUNEL 实验可以再次证明在栉孔扇贝血淋巴细胞中存在高渗透压引发 DNA 断裂，低渗透压诱导 DNA 修复的现象。高渗透压贝产生的大量 DNA 断裂对于栉孔扇贝有什么生物学意义呢？扇贝 长期生活在海水中，这些 DNA 断裂又会不会被修复呢？这些都是下一步有待研究 的问题。关键词：栉孔扇贝；原代培养；彗星实验；TUNEL 实验Study on the Relationship between Chlamys farreri’s DNA Breaks and Seawater Osmolarity PressureAbstractNot a lot of studies have already been made in the osmotic effects on terrestrial life’ DNA, let alone marine life, so this research is in the frontier of related studies. In this research, Comet Assay and TUNEL assay are used in the primary Chlamys farreri hemolymph culture, and a phenomenon has been shown that high osmotic pressure of the seawater (1200mOsm/L) will cause Chlamys farreri’s DNA fragmentation, producing 3-OH ends. And this DNA fragmentation is reversible when the osmolarity of external environment is adjusted to the low osmolarity levels (460mOsm/L) , when DNA breaks will be automatically repaired quickly.Primary Chlamys farreri Blood Cells Culture.Chlamys farreri blood cells is used for primary culture. The improved L-15 medium of 1200mOsm/L is uesd as basic medium, and cuture are under 20 ℃, pH = 7.6. Cultured blood cells within 1~3 days will adhere to petri dish widely, and the blood cells’ viability rate is higher than 90%. After 10~15 days’ culturing, the cells gradually die . The first 3 days’ cells in culture fully meet the Comet Assay TUNEL assay’s standards.Comet AssayComet Assay is carried on the base of primary Chlamys farreri blood cell culture, and use logarithmic growth phase H1299 cells as negative control. It is found after 32h continuous monitoring that, blood cells in low osmolality (460mOsm/L) always have a much lower Tail DNA% than the ones in high osmolarity (1200mOsm/L) , the former is in the range of 6.04% ~ 13.29% , and the latter gradually increase from 28.75% to 60.86%. After the exch