The Function And Mechanism Of Exogenous CCN1 In Cell Damaged By Radiation

ObjectiveIt is easy to generate nuclear radiation induced injury in normal time or wartime. However, as the injury affiliated with nuclear radiation induced injury healed slowly or even can't cure, the mechanism of which needs further investigaiton. Collagenoblasts are the most cells in connective tissue proper, and are the major cells involved in wound healing. The fibroblasts injury certainly affects the progression of granulation tissue generation and other tissue repair, which may be one of the most important causes why it is difficult to cure the nuclear radiation induced injury. Cyr61/CCN1 is a member of CCN protein family,which can promote the cell proliferation, adhension and differentiation,and plays an important role in extracellular matrix formation. However, the relationship between CCN1 and radiation induced injury, and the detailed mechanism is still unknown. This study is to detect the expression stutus of mouse fibroblasts L929 induced by radiation, using CCN1 conditional medium to culture radiation induced cells, and observe the regulation of CCN1 on the factors related with growth, migration and wound healing. According to the sdudy, we will get approach to the function and mechanism of CCN1 towards the repair of cells injured by radiation, and shed light on the mechanism of wound healing and new drug for healing.Method:1. Mouse fibroblasts are exposed in different does of irradiance (2, 4, 6, 8Gy) with 60Coγray (2, 4, 6, 8Gy). Cell survival scores are calculated and cell survival curve is drawn, and the optimization dose inducing L929 cells injury is determined.2. L929 cells are exposed in the optimization dose of 4 Gy 60Coγray, using MTT and Clonogenic Assay to detect the influence of radiation on cells growth. DNA ladder electrophoresis method and TUNEL test are used to detect the radiation induced apoptosis, and flow cytometry is used to detect the cell cycle. 3. ICC and RT-PCR are used to detect 4 Gy60Coγray induced CCN1 expression alteration at different time(0, 6, 12, 24, 48, 72h) in L929 cells, and RT-PCR is used to detect expression of CCN1 in different irradiance(1,5,10Gy) after 24h.4. HEK293 cells are used to amplify RFP marked AdCCN1 and control AdRFP, and make conditional medium: using AdCCN1 and AdRFP to respectively infect CHO cells, detect the RFP expression 16h later under the fluorescence microscope, continue to culture in medium with low blood serum, collect the supernatant 48h later, and freeze, dry and condense, and at last use Western blot to characterize the protein expression.5. CCN1 and RFP conditional medium are used respectively to culture radiation induced L929 cells, using MTT and Clonogenic Assay to detect cell growth curve and exogenous CCN1 cloning efficiency of radiation induced L929 cells cultured with exogenous CCN1. Scratch-cure test and flow cytometry are used to respectively detect the migration and cell cycle alteration of L929 cells.6. RT-PCR is used to detect mRNA level of ColⅠ, MMP1, TTMP1 in radiation injured L929 cells cultured in conditional medium for(1, 3, 5 d), and ICC is used to detect their protein levels at 5 d.Results1. All dose of 60Coγradiation will affect the L929 cells'survival, and according to the cell survival curve the D37 dose is 4 Gy, so 4 Gy 60Coγray is selected to perform the following test.2. 60Coγray's influence on cell growth①2d after exposed by 4 Gy 60Coγray, the proliferation of L929 is significantly inhibited. Compared with the control group, the clone formation and cloning efficiency of radiation group is significantly lower.②Ladder spectra can't be seen in DNA ladder electrophoresis method, and positive cells can't be seen by TUNEL staining.③Distribution of cell cycle in different group: after radiation for 24h, cells in G2/M increases 6.01 times, cells in S decreases 7.06 times, and cells in G0/G1 decreases 1.26 times, which indicates that radiation can result in the L929 cell cycle arrest in G2.3. 60Coγray's influence on CCN1 expression in L929 cellsResults of ICC and RT-PCR: 24, 48h after radiation of 60Coγray, the radiation group expresses significantly less mRNA of CCN1 than the control group(P<0.01), 6h after radiation, the mRNA of CCN1 becomes less, and 24h later,the level begins to increase. Compared to control group,all the radiation groups(1G, 5Gy, 10Gy) have lower CCN1 mRNA level(P<0.05), and the decreased mRNA level appears radiation dose dependent.4. Maked CCN1 and RFP conditional medium can be seen fluorescence under fluorescence microscope, and the CCN1 conditional medium contains CCN1 protein confirmed by western blot, but RFP conditional medium doesn't.5. Regulation of exogenous CCN1 to L929 cells injured by radiation①CCN1 significantly promote cell proliferation. CCN1 group is higher than RFP group in clone formation and cloning efficiency.②Cure rate of scratched injury: CCN1 group cures better than RFP group(P<0.01) at 1~5 d, such as cure rate of 1d: CCN1 group(18.73±5.33)%, RFP group (12.11±4.03)%; 3d: CCN1 group(74.48±9.19)%,RFP group(54.38±6.57)%.③Distribution of cell cycle in L929 cells: Compared with RFP group, after 5d, stage S with CCN1 group cells increases 1.48 times, and stage G2/M decreases 1.73 times, which indicates that CNN1 can promote caryocinesia, and promote cell proliferation.6. Regulation of exogenous CCN1 to wound healing correlation factors in L929 cells injured by radiation: L929 cells injured by radiation are treated with CCN1 conditional medium for 1d, and the mRNA level of MMP1 and TIMP1 are lower compared with RFP group, and ColⅠis higher (P<0.01); 3d later, the MMP1 and TIMP1 in CCN1 group is higher than RFP group, but there is no statistical significance. ColⅠis lower in CCN1 group than in RFP group(P<0.01); 5d later, CCN1 group has higher MD of MMP1 positive cells(P<0.05), and the expression of TIMP1 increases, but has lower MD of MMP1 positive cells than RFP group(P<0.05).Conclusion1. Exposing L929 cells in 4 Gy 60Coγray can inhibit cell proliferation, decrease clone formation, but have no apoptosis. The cell cycle arrests in stage G2, and interrupts the normal caryocinesia, and finally inhibits the cell prolieration. Protein and mRNA level of CCN1 decreases in radiation induced L929 cells in a dose dependent manner. These results indicate that the expression of CCN1 may be related to the radiation induced cell proliferation.2. CCN1 can promote the proliferation and migration of radiation induced cells, and increases the cells in stage S, which indicates that CCN1 may have function of promoting wound healing , and that low expression of CCN1 may account for the poor repair status of wound accompany with radiation.3. CCN1 can upregulates the expression of wound healing correlation factor MMP1 in a certain period and downregulates ColⅠsignifican. These results indicate that CCN1 gene can regulate the expression of some wound healing correlation factors, and play an improtant role in promoting radiation injuries.