Establish Stable Expression GFP-α-tubulin/GFP-CENP-A-HeLa Cell Line And Evaluate Its Function

The cancer,one of the chronic non-communicable diseases, has become a hazard to human health and cause of human death, and WHO released the latest report shows that malignant cancer incidence and mortality still showing rapid growth trend. Therefore, it is very urgent to clarify the reason arising cancer from the cellular and molecular leve l, which will shed light on the cancer treatment and prevention. With the recent development of molecular imaging, high-resolution, high-speed live cell imaging microscope system; we can study the mechanism of tumor progression from the cellular level. In order to apply this technology to achieve the goal of screening effective chemotherapy treatment of tumors, it is urgent to build a assay to evaluate whether chemotherapy drugs kill the tumor cells. Therefore we construct the stable cell line expressed microtubule and kinetochore associated fluorescent proteins to observe the behavior of tumor cells under chemotherapy drugs treatment, which providing a powerful tool for cancer therapy and screening of chemotherapy drugs.Defective regulate the cell cycle ca n lead to tumorigenesis. Regulation of mitosis is afine-tuning process in which each pair of kinetochores must be properly attached with microtubule from spindle pole. Defection of kinetochore- microtubule attachment can lead misalignment of chromosomes, which will led to abnormal chromosome segregation and promote chromosomal instability and aneuploidy, In order to detailed observe binding process of chromosome and microtubule, a green fluorescent protein(GFP) labeled microtubules and kinetochore-associated proteins in tumor cell line was established. We want to study the effect of different chemotherapeutic drugs though live-cell microscopy workstation and this will promote further study of tumorigenesis and cancer treatment.Objective: 1. The study was designed to build a tumor cell line model with green fluorescent protein(GFP) labeled microtubules and kinetochore-associated proteins. 2. The study was designed to evaluate the effect of microtubule-associated chemotherapeutic drugs in cell cycle, though GFP-CENP-A /α-tubulin-He La cell line.Methods: 1. Molecular cloning of the plasmids for lentiviral production, and obtain the lentivirus from supernatant of 293 T cell for infection. 2. Establishment of cell synchronization assay to obtain cells from G1 phase by adding thymidine and release cel s to mitosis. 3. Using time-lapse live cell imaging workstations to observe mitotic procedure of cells. 4. The cells were treated with Nocodazole and Taxol to observe the dynamic changes of apoptosis and change of microtubule polymerization.Results:1. Though molecular clone technique, we successfully inserted C ENP-A /α-tubulin fragment into the lentiviral vector with GFP-tag. Then we produced lentivirus by 293 T cel s and ultimately got He La cell line stably expressing GFP-CENP-A / α-tubulin. 2. By confocal microscopy, we observed microtubules with green fluorescence in interphase cel s, that suggest this cell line can be used for live-cell imaging.. 3. Establish a synchronization method based on chemical drugs, we successfully synchronized He La cells in G1 phase and then got cells in G1/S, S, G2/M phase through releasing cel s into normal medium. 4. Using Time- Lapse living cells workstation, we successfully observed the dynamic process of microtubules and kinetochores attachment by GFP-CENP-A /α-tubulin-He La cell line synchronizing in mitosis. 5. Though comprising mitotic progression of GFP-CENP-A/α-tubulin-He La cells with normal cells, suggesting that the two cell line had no significant difference in the time of cell division. So this cell line can be used for further study. 6. In order to study the mechanism of microtubule associated chemotherapeutic drug, we take advantage of Time-Lapse workstation to dynamically observe the process of microtubule re- growth after withdrawal of the drug. The results showed GFP-CENP-A/α-tubulin have been stably integrated in genome of He La cells, and this cell line can be used as a powerful tool for research the mechanism of microtubule associated chemotherapeutic drug.ConclusionWe successfully obtain the stable cell line expressing GFP-CENP-A(K inetochore) and GFP-α-tubulin(microtubules) by lentivirus infection, and we successfully observed attachment procedure of kinetochore-microtubules in mitosis. Though treatment by microtubule poison, we found the cell line can be used for evaluating the function of microtubule in cancer cells under chemotherapeutic agent treatment. The successful establishment of this cell line will supply a good model for understanding mechanism ofkinetochore-microtubule attachment and cancer therapy.