Huang, Fei
(Department of Pathology, The Affiliated Tumor Hospital Of Xinjiang Medical University)
,
Zhao, Feng
(Department of Pathology, The Affiliated Tumor Hospital Of Xinjiang Medical University)
,
Liang, Li-Ping
(Department of Pathology, The Affiliated Tumor Hospital Of Xinjiang Medical University)
,
Zhou, Mei
(Department of Pathology, The Affiliated Tumor Hospital Of Xinjiang Medical University)
,
Qu, Zhi-Ling
(Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology)
,
Cao, Yan-Zhen
(Department of Pathology, The Affiliated Tumor Hospital Of Xinjiang Medical University)
,
Lin, Chen
(Department of Pathology, College of Basic Medicine, Xinjiang Medical University)
Background: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfec...
Background: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Materials and Methods: $Lipofectamine^{TM}2000$ as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA, miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. Results: The seeding density of Hela cell line and Siha are $1.5{\times}10^4$ (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01). MTT assay showed that according to the above conditions which has the lowest cytotoxicity. Conclusions: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.
Background: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Materials and Methods: $Lipofectamine^{TM}2000$ as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA, miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. Results: The seeding density of Hela cell line and Siha are $1.5{\times}10^4$ (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01). MTT assay showed that according to the above conditions which has the lowest cytotoxicity. Conclusions: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.
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문제 정의
The objectives of this study are to explore the feasibility of liposome transfected cervical cancer cells, obtain the optimal transfection conditions of liposome transfected cervical cancer cells, use it to establish liposome transfection mediated of cervical cancer cell system.
가설 설정
2) The formation of DNA / liposome complexes can not limited by the size of DNA.3)Having a higher transport capacity for DNA which with a high charge.
제안 방법
The experiment we used Hela cell line and Siha cell line, from seeding density, the amount of miRNA and its proportion of liposomes, transfection time, the presence or absence of serum and cell toxicity experiments etc to explore how to improve the efficiency of transfection of cervical cancer cells line. It can be suggested that Liposomes is an appropriate reagent which use miRNA to transfect into Hela cell and Siha cell, and the rate of expression of a candidate gene can be evaluated easily after transfection by using this reagent.
이론/모형
The MTT method was used to estimate cell viability. We use the optimal transfection conditions of the above experiments to MTT assay.
성능/효과
After 24 hours transfection, observed, the results show that when the amount of miRNA is 0.5 ul, Obviously there is not has a high transfection efficiency; while, When the amount of miRNA is 1ul or 1.25ul, the results of transfection do not change substantially. This shows that when cells were seeded at the same density, after achieve the optimum amount of miRNA, The efficiency of transfection no longer following the amount of miRNA to increases, excessive amount of miRNA will lead to a waste of reagents.
In this experiment where LipofectamineTM2000 amount unchanged, The transfection efficiency of showed a trend of increase with the increase of miRNA dosage. However, when the quantity of miRNA more than 1ul in per well of 24-well plate ,the transfection efficiency is in the doldrums.
The experimental results show that when the reaction time of miRNA/ liposomes compounds is less than 6 hours, the transfection efficiency is low because it haven’t fully embodying the exogenous gene of the cell.
No serum before transfection group than those with serum group has a high transfection efficiency only exist in Siha cell. There were significant differences (P[0.01); After 6 hours for transfection, the efficiency of transfection is not statistically significant difference between no serum group and serum group (P]0.05). But for Hela, there was no statistically significant difference between two groups of results.
후속연구
It can be suggested that Liposomes is an appropriate reagent which use miRNA to transfect into Hela cell and Siha cell, and the rate of expression of a candidate gene can be evaluated easily after transfection by using this reagent. Through this experiment, it can help to provide assistance for further research which in miRNA regulatory mechanism in cervical cancer in the future.
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