山东医药
山東醫藥
산동의약
SHANDONG MEDICAL JOURNAL
2014年
35期
23-26
,共4页
磷酸钙转染法%细胞转染%细胞存活率%细胞状态
燐痠鈣轉染法%細胞轉染%細胞存活率%細胞狀態
린산개전염법%세포전염%세포존활솔%세포상태
calcium phosphate transfection method%cell transfection%cell viability%the state of cell
目的：优选出pMaxGFP导入人肝癌HepG2细胞中磷酸钙转染法的应用条件。方法取对数生长期的人肝癌HepG2细胞，采用磷酸钙转染法将pMaxGFP导入HepG2细胞，转染前1 h换不同血清浓度的新鲜培养液培养HepG2细胞，血清浓度分别为无血清、1％、2％、5％、10％、15％。转染48 h后以倒置荧光显微镜观察绿色荧光蛋白表达，评估转染效率，同时以原位台盼蓝染色检测转染后的细胞存活率，优选出最适血清浓度。在最适血清浓度条件下，磷酸钙转染法将pMaxGFP转染贴壁状态和悬浮状态的HepG2细胞，共分为A、B、C、D、E 5组，分别代表转染贴壁状态、悬浮状态3×104细胞/孔、4×104细胞/孔、5×104细胞/孔、6×104细胞/孔。转染48 h后在荧光倒置显微镜下观察绿色荧光蛋白表达，评估转染效率，同时以原位台盼蓝染色检测转染后的细胞存活率，优选出最适的细胞状态和细胞密度。结果48h后，无血清组HepG2细胞大量死亡，而其他组有较少部分细胞死亡。1％、2％、5％、10％、15％血清浓度的转染效率分别为30．92％±1．29％、30．10％±1．05％、21．27％±0．63％、19．10％±0．51％、10．44％±1．42％，血清浓度为1％、2％时的转染效率明显高于其他血清浓度，P＜0．05；1％、2％、5％、10％、15％血清浓度的细胞存活率分别为76．01％±0．94％、89．68％±0．68％、92．38％±1．02％、93．36％±1．35％、93．49％±0．91％，血清浓度为2％时的细胞存活率明显高于1％血清浓度。在转染培养基血清浓度为2％条件下， A、B、C、D、E组转染效率分别为30．74％±0．50％、6．38％±0．28％、31．41％±0．42％、30．94％±0．47％、1．41％±0．27％， A、C、D组转染效率明显高于B、E组，P﹤0．05；A、B、C、D、E组细胞存活率分别为91．76％±0．99％、88．40％±1．29％、88．38％±1．08％、87．48％±0．78％、64．45％±0．55％，A、B、C、D组细胞存活率明显高于E组。结论优选出pMaxGFP导入人肝癌HepG2细胞中的磷酸钙转染法应用条件为转染培养基中血清浓度2％、悬浮状态下细胞密度为4×104细胞/孔或5×104细胞/孔。
目的：優選齣pMaxGFP導入人肝癌HepG2細胞中燐痠鈣轉染法的應用條件。方法取對數生長期的人肝癌HepG2細胞，採用燐痠鈣轉染法將pMaxGFP導入HepG2細胞，轉染前1 h換不同血清濃度的新鮮培養液培養HepG2細胞，血清濃度分彆為無血清、1％、2％、5％、10％、15％。轉染48 h後以倒置熒光顯微鏡觀察綠色熒光蛋白錶達，評估轉染效率，同時以原位檯盼藍染色檢測轉染後的細胞存活率，優選齣最適血清濃度。在最適血清濃度條件下，燐痠鈣轉染法將pMaxGFP轉染貼壁狀態和懸浮狀態的HepG2細胞，共分為A、B、C、D、E 5組，分彆代錶轉染貼壁狀態、懸浮狀態3×104細胞/孔、4×104細胞/孔、5×104細胞/孔、6×104細胞/孔。轉染48 h後在熒光倒置顯微鏡下觀察綠色熒光蛋白錶達，評估轉染效率，同時以原位檯盼藍染色檢測轉染後的細胞存活率，優選齣最適的細胞狀態和細胞密度。結果48h後，無血清組HepG2細胞大量死亡，而其他組有較少部分細胞死亡。1％、2％、5％、10％、15％血清濃度的轉染效率分彆為30．92％±1．29％、30．10％±1．05％、21．27％±0．63％、19．10％±0．51％、10．44％±1．42％，血清濃度為1％、2％時的轉染效率明顯高于其他血清濃度，P＜0．05；1％、2％、5％、10％、15％血清濃度的細胞存活率分彆為76．01％±0．94％、89．68％±0．68％、92．38％±1．02％、93．36％±1．35％、93．49％±0．91％，血清濃度為2％時的細胞存活率明顯高于1％血清濃度。在轉染培養基血清濃度為2％條件下， A、B、C、D、E組轉染效率分彆為30．74％±0．50％、6．38％±0．28％、31．41％±0．42％、30．94％±0．47％、1．41％±0．27％， A、C、D組轉染效率明顯高于B、E組，P﹤0．05；A、B、C、D、E組細胞存活率分彆為91．76％±0．99％、88．40％±1．29％、88．38％±1．08％、87．48％±0．78％、64．45％±0．55％，A、B、C、D組細胞存活率明顯高于E組。結論優選齣pMaxGFP導入人肝癌HepG2細胞中的燐痠鈣轉染法應用條件為轉染培養基中血清濃度2％、懸浮狀態下細胞密度為4×104細胞/孔或5×104細胞/孔。
목적：우선출pMaxGFP도입인간암HepG2세포중린산개전염법적응용조건。방법취대수생장기적인간암HepG2세포，채용린산개전염법장pMaxGFP도입HepG2세포，전염전1 h환불동혈청농도적신선배양액배양HepG2세포，혈청농도분별위무혈청、1％、2％、5％、10％、15％。전염48 h후이도치형광현미경관찰록색형광단백표체，평고전염효솔，동시이원위태반람염색검측전염후적세포존활솔，우선출최괄혈청농도。재최괄혈청농도조건하，린산개전염법장pMaxGFP전염첩벽상태화현부상태적HepG2세포，공분위A、B、C、D、E 5조，분별대표전염첩벽상태、현부상태3×104세포/공、4×104세포/공、5×104세포/공、6×104세포/공。전염48 h후재형광도치현미경하관찰록색형광단백표체，평고전염효솔，동시이원위태반람염색검측전염후적세포존활솔，우선출최괄적세포상태화세포밀도。결과48h후，무혈청조HepG2세포대량사망，이기타조유교소부분세포사망。1％、2％、5％、10％、15％혈청농도적전염효솔분별위30．92％±1．29％、30．10％±1．05％、21．27％±0．63％、19．10％±0．51％、10．44％±1．42％，혈청농도위1％、2％시적전염효솔명현고우기타혈청농도，P＜0．05；1％、2％、5％、10％、15％혈청농도적세포존활솔분별위76．01％±0．94％、89．68％±0．68％、92．38％±1．02％、93．36％±1．35％、93．49％±0．91％，혈청농도위2％시적세포존활솔명현고우1％혈청농도。재전염배양기혈청농도위2％조건하， A、B、C、D、E조전염효솔분별위30．74％±0．50％、6．38％±0．28％、31．41％±0．42％、30．94％±0．47％、1．41％±0．27％， A、C、D조전염효솔명현고우B、E조，P﹤0．05；A、B、C、D、E조세포존활솔분별위91．76％±0．99％、88．40％±1．29％、88．38％±1．08％、87．48％±0．78％、64．45％±0．55％，A、B、C、D조세포존활솔명현고우E조。결론우선출pMaxGFP도입인간암HepG2세포중적린산개전염법응용조건위전염배양기중혈청농도2％、현부상태하세포밀도위4×104세포/공혹5×104세포/공。
Objective To optimize the application conditions of calcium phosphate transfection method when pMaxG-FP is delivered to human hepatoma HepG2 cells.Methods pMaxGFP was delivered to human hepatoma HepG2 cells of logarithmic growth phase by calcium phosphate transfection method.1 h before transfection, different serum concentrations of fresh medium which were null, 1%, 2%, 5%, 10%and 15%were changed to every culture wells.After 48 h, invert-ed fluorescence microscope was used to observe the expression of GFP to evaluate the transfection efficiency, while in situ try pan blue staining to detect cell viability.Thus, the optimal serum concentration was found.Then, under the condition of optimal serum concentration, the following experiment was divided into A, B, C, D, E five groups, namely adherent state, suspended 3 ×104 cells /well, 4 ×104 cells /well, 5 ×104 cells /well, 6 ×104 cells /well.After 48 h, inverted fluorescence microscope was used to observe the expression of GFP to evaluate the transfection efficiency, while in situ try pan blue staining to detect cell viability.Then the best state of cell and its density were optimized in the same way.Results After 48 h, there were a great number of dead cells in the serum-free group, while a little in the other group.In the 1%,2%, 5%, 10%, 15% serum concentration group, transfection efficiency was 30.92%±1.29%, 30.10%±1.05%, 21.27%±0.63%, 19.10%±0.51%, 10.44%±1.42%, respectively.Transfection efficiency was significantly higher than the other groups when the serum concentration was 1%and 2%, P<0.05;in the 1%, 2%, 5%, 10%, 15%serum concentration group, cell viability was 76.01%±0.94%, 89.68%±0.68%, 92.38%±1.02%, 93.36%±1.35%, 93.49%±0.91%, respectively.Cell viability in the 1%serum concentration group was significantly higher than the 2%. Under the condition of 2%serum concentration, transfection efficiency of A, B, C, D, E group was 30.74%±0.50%, 6.38%±0.28%, 31.41%±0.42%, 30.94%±0.47%, 1.41%±0.27%, respectively.A, C and D groups were sig-nificantly higher than B and E groups, P<0.05;cell viability of A, B, C, D, E group was 91.76% ±0.99%, 88.40%±1.29%, 88.38% ±1.08%, 87.48%±0.78%, 64.45% ±0.55%, respectively.A, B, C and D groups were sig-nificantly higher than E group.Conclusion The application conditions of calcium phosphate transfection method when pMaxGFP is delivered to human hepatoma HepG2 cells are 2%serum concentration and 4 ×104 cells/well or 5 ×104 cells/well when suspending transfection.
