Preliminary Study Of Ca²⁺/CaM/CaMKâ…¡ Signaling Pathway In Rat Liver Regeneration

Partt ⅠIsolation, culture and identification of hepatocyte in neonatal ratsObjective:To establish a simple and effective isolation and culture method of hepatocyte in neonatal rats.Method:The neonatal liver cell was collected with Collagenase-tissue explants digestion method after a multi-step low-speed centrifuge.The RT-PCR and Western blot techniques were adopted to analyse the mRNA and protein expression of ALB、 CK-18、Ck-8 and AFP when the hepatocyte in vitro had partially growed to cell colony. These cells’biological function was identified and compared with liver tissue of congener neonatal rats and mature rats.Result:The cultured liver cell of neonatal rats was isolated and was round in shape under inverted microscope. After 24 hours, part of cells adhered to the wall with island-like stretched cell body which had turned to oval from round. After 48 hours, cells came to proliferation period with the character of epithelial cells—irregular polygon. After 3 days, the cultured hepatocytes connected each other into an integrity with reduced transparency and clear profiles cell body; parts of liver cells had double or multiple nucleus. After 5 days, the cells were basically the same in shape with clone-like growth and integrity with each other. RT-PCR technique detected the mature liver cell marker ALB、CK-18、CK-8 mRNA which expressed by the hepatic cells in neonatal rats; Western blot technique examined the mature hepatocyte marker ALB、CK-18、CK-8 protein of the liver cells in the neonatal rats.Conclusion:The hepatocytes in neonatal rats are isolated and identified successfully and able to be cultured in vitro.Result:The cultured liver cell of neonatal rats was isolated and was round in shape under inverted microscope. After 24 hours, part of cells adhered to the wall with island-like stretched cell body which had turned to oval from round. After 48 hours, cells came to proliferation period with the character of epithelial cells—irregular polygon. After 3 days, the cultured hepatocytes connected each other into an integrity with reduced transparency and clear profiles cell body; parts of liver cells had double or multiple nucleus. After 5 days, the cells were basically the same in shape with clone-like growth and integrity with each other. RT-PCR technique detected the mature liver cell marker ALB、CK-18、CK-8 mRNA which expressed by the hepatic cells in neonatal rats; Western blot technique examined the mature hepatocyte marker ALB、CK-18、CK-8 protein of the liver cells in the neonatal rats.Conclusion:The hepatocytes in neonatal rats are isolated and identified successfully and able to be cultured in vitro.Part ⅡPackaging, titration, screening and identification of CaMKⅡγ gene lentvirusObjective:To explore the over-expression and interference of CaMK II y gene induced by the lentvirus system in rat liver cells. To lay a foundation for the further researching the mechanism of CaMK Ⅱγ gene in liver regeneration.Method:The over-expression vector and over-expression empty vector were constructed based on rat mRNA sequence of CaMK Ⅱγ gene and synthetic CaMKⅡγygene.Three pairs of shRNA interference arrays and one pair of control array were designed, three shRNA interference lentvirus vectors and interference empty vector were constructed in the same time and transferred into Stb13 cells to sequence, then transferred into 293Ta cells induced by liposome and packaged to lentvirus with titration test. Then different lentvirus were transferred into rat liver cells respectively and grouped to CON group(non-infection cell group),LV-CaMKⅡγ group(infected by lentvirus over-expression vector CaMK Ⅱγ group), LV-NC(infected by lentvirus over-expression empty vector group), shRNA-1 group(infected by lentvirus interference vector CaMKⅡγ-shRNA-1 group), shRNA-2 group (infected by lentvirus interference vector CaMKⅡγ- shRNA-2 group),shRNA-3 group (infected by lentvirus interference vector CaMK II y-shRNA-3 group), shRNA-NC group (infected by lentvirus interference empty vector group).The Western blot and RT-PCR techniques were used to examine the expression of CaMKⅡγmRNA and protein in each groups so that the feasibility of one pair of shRNA which had more effective over-expression and interference efficiency on CaMKⅡγ was ensured.Result:①The lentvirus CaMK Ⅱγ over-expression vector, interference vector were established and lentvirus was packaged successfully. ②The virus titer of CaMK II y over-expression vector,CaMK Ⅱγ over-expression empty vector, CaMKⅡγ-shRNA-1 vector, CaMKⅡγ-shRNA-2, CaMK Ⅱγ-shRNA-3, CaMK Ⅱγ-shRNA-NC were tested by RT-PCR:3.86×108copies/ml,1.26×108copies/ml,9.54×108copies/ml, 8.32×108copies/ml,9.04×108copies/ml,2.28×108copies/ml. ③The rat liver cells were infected by each group of lentvirus successfully.④The result of expression of CaMKⅡγ mRNA:48 hours after infection, the CaMKⅡγ mRNA relative content (CaMKⅡγ/GAPDH) in cells of CON,LV-CaMK Ⅱγ,LV-NC groups separately were 1.000,10.679±0.021,0.968±0.010, the content of CaMKⅡγ mRNA in LV-CaMKⅡγ group up-regulated apparently compared with CON and LV-NC group, with statistical significance(P<0.001).the LV-CaMKⅡγ group showed a better over-expression effect;48 hours after infection, the CaMKⅡγ mRNA relative content (CaMKⅡγ/GAPDH) in cells of CON, shRNA-1,shRNA-2,shRNA-3,shRNA-NC groups separately were 1.000,0.114±0.005,0.457±0.008,0.561±0.006,1.062±0.007, the content of CaMK II y mRNA in shRNA-1, shRNA-2, shRNA-3 group were down-regulated apparently compared with CON and LV-NC group. The interference effect of shRNA-1 was remarkably higher than shRNA-2 and shRNA-3 group, with statistical significance(P<0.001).the interference efficiency of each group were: (85.6±0.5)%, (54.3±0.8)%, (43.9±0.6)%, shRNA-1 group exhibited a better interferece effect, ⑤the semi-quantity result of CaMK Ⅱγ protein expression tested by the Western blot:48 hours after infection, the expression of LV-CaMKⅡγ group was obviously higher than CON and LV-NC group with over-expression of CaMKⅡγ, with statistical significance(P<0.05).the expression of shRNA-1 was obviously lower than CON,shRNA-2,shRNA-3 and shRNA-NC group with silence of CaMK Ⅱγ, with statistical significance(P<0.05).Conclusion:The CaMK Ⅱγ gene over-expression technique induced by lentvirus is constructed successfully in rat hepatocytes and has achieved over-expression of gene; The silence of CaMK Ⅱγ gene with the interference lentvirus in rat liver cells is established successfully and confirms the apparent down-regulation of target gene’s expression level, lays a foundation on follow-up research that the influence by over-expression and silence of CaMK Ⅱγ gene as the target gene to liver regeneration and provides reliable theory basis for in vitro study of liver regeneration. (CaMKⅡγ/GAPDH) in cells of CON, shRNA-1,shRNA-2,shRNA-3,shRNA-NC groups separately were 1.000,0.114±0.005,0.457±0.008,0.561±0.006,1.062±0.007, the content of CaMK II y mRNA in shRNA-1, shRNA-2, shRNA-3 group were down-regulated apparently compared with CON and LV-NC group. The interference effect of shRNA-1 was remarkably higher than shRNA-2 and shRNA-3 group, with statistical significance(P<0.001).the interference efficiency of each group were: (85.6±0.5)%, (54.3±0.8)%, (43.9±0.6)%, shRNA-1 group exhibited a better interferece effect, ⑤the semi-quantity result of CaMK Ⅱγ protein expression tested by the Western blot:48 hours after infection, the expression of LV-CaMKⅡγ group was obviously higher than CON and LV-NC group with over-expression of CaMKⅡγ, with statistical significance(P<0.05).the expression of shRNA-1 was obviously lower than CON,shRNA-2,shRNA-3 and shRNA-NC group with silence of CaMK Ⅱγ, with statistical significance(P<0.05).Conclusion:The CaMK Ⅱγ gene over-expression technique induced by lentvirus is constructed successfully in rat hepatocytes and has achieved over-expression of gene; The silence of CaMK Ⅱγ gene with the interference lentvirus in rat liver cells is established successfully and confirms the apparent down-regulation of target gene’s expression level, lays a foundation on follow-up research that the influence by over-expression and silence of CaMK Ⅱγ gene as the target gene to liver regeneration and provides reliable theory basis for in vitro study of liver regeneration.Part ⅢPreliminary study of Ca2+/CaM/CaMK Ⅱ signaling pathway in liver regeneration after partial hepatectomyObjective:To explore the impact of Ca2+/CaM/CaMK Ⅱ signaling pathway in liver regeneration after partial hepatectomy,and to observe the changes in liver regeneration after partial hepatectomy with injecting the CaMK Ⅱγ gene into rats so that to study the changes in Ca2+/CaM/CaMK Ⅱ signaling pathway in liver regeneration after partial hepatectomy initially and to lay a foundation for the study of advanced liver regeneration.Method:(1 Establishment of animal model:the SD rat models of 60% partial Hepatectomies were stably constructed by the hepatic left,dual-caudate, right lower and right higher lobectomy.(2)experiment grouping:there were 5 groups after partial hepatectomy, A group(normal saline control group),B group(infected by lentvirus over-expression empty vector group),C group(infected by lentvirus over-expression vector CaMK Ⅱγ group),D group(infected by lentvirus interference empty vector group), E group(infected by lentvirus interference vector CaMKⅡγ-shRNA group). They were all dosed by injection through the appendix vein in operation. The dosage of lentvirus was 1.0×10 TU/rat and the normal saline control group was injected tha same volume of normal saline 500μl. the blood sample and tissue specimen were collected at 1,3,5 and 7 days postoperative in all groups.(3)index of correlation examination: ①to test the index of liver regeneration; ②to measure the changes of liver function indexes by the automatic biochemistry analyzer; ③to detect the changes of Ca2+ by the flow cytometry technique after Ca2+was marked by Fluo4-AM fluorescent probe;④to test the content of CaM and CaMK Ⅱγ mRNA by the RT-PCR technique in all groups; ⑤to examine the expression of CaM and CaMK Ⅱγ protein by the western blot technique in all groups; ⑥to detect the expression of CaM and CaMK Ⅱγ protein by the immunohistochemical technique in all groups.Result:(1) SD rat models of 60% partial Hepatectomies were constructed successfully through plenty of surgery operation training.(2) the liver cells were infected successfully by the lentvirus of every group in the rats.(3)the results of index of correlation test: ①the index of liver regeneration:the percentage of the residual liver of all groups increased with time. There was no significant difference between all groups at 1 day. C group was obviously higher than A group (P<0.05),and A group was obviously higher than E group(.P<0.05),however, there was no significant difference between A,B and D group at 3 and 5 days. E group was obviously lower than A group (P<0.05),and here was no significant difference between A,B,C and D group at 7 days, ②the results of liver function:the level of ALT and AST of all groups reached a peak at 1 day postoperative and showed a downtrend with time, with statistical significance(P<0.05). the level of ALT and AST of C and E group was higher than that of A group(P<0.05). There was no significant difference between C and E group, and there was no significant difference between A,B and D group. There was no significant difference between the 5 groups at 3,5 and 7 days. The level of TBIL reached a peak at 1 day postoperative, and then showed a downtrend with time, with statistical significance(P<0.05). the level of TBIL at 1,3,5 and 7 days in of alls groups had no significant difference (P> 0.05).③the Ca2+ tested by the flow cytometry:the differences of the Ca2+proportion in liver cells between every group and every time point postoperative had statistical significance and the Ca2+ proportion in liver cells reached the highest peak at 1 day postoperative, and then showed a downtrend with time and increased at 7 days. There was no significant difference of the Ca2+proportion at 1 day postoperative between all time points and all groups. At 3 days, E group was obviously higher than A group (P<0.05), and A group was obviously higher than C group(P<0.05),and there was no significant difference between A,B and D group. C group was obviously higher than A group at 5 and 7 days (P<0.05),and A group was obviously higher than E group(P<0.05),and there was no significant difference between A,B and D group.④ the expression results of CaM and CaMKⅡmRNA tested by RT-PCR:the differences of CaM and CaMK Ⅱ mRNA between all groups and all time points had statistical significance and increased with time. There was no significant difference of CaM and CaMK Ⅱ mRNA at 1 day postoperative between all time points and groups. C group was obviously higher than A group at 3,5 and 7 days(P<0.05),and A group was obviously higher than E group(P<0.05),however, there was no significant difference between A,B and D group. ⑤ the expression results of CaM and CaMK Ⅱ protein tested by Western blot:the differences of CaM and CaMK Ⅱ protein between all groups and all time points had statistical significance and increased with time. There was no significant difference of CaM and CaMK Ⅱ protein at 1 day postoperative between all time points and groups. C group was obviously higher than A group at 3,5 and 7 days(P< 0.05),and A group was obviously higher than E group(P<0.05),however, there was no significant difference between A,B and D group.⑥the expression results of CaM and CaMK Ⅱ protein tested by immunohistochemical technique:the differences of CaM and CaMK Ⅱ protein between all groups and all time points had statistical significance and the positive rate increased with time. The contrast results of CaM and CaMK Ⅱ between all time points and groups showed than:There was no significant difference at 1 day postoperative between all groups. The positive rate of C group was the highest while that of E group was the lowest at 3,5 and 7 days. C group was obviously higher than A group(P<0.05),and A group was obviously higher than E group(P<0.05),however, there was no significant difference between A,B and D group. Conclusion:There are some regulating effect of Ca2+/CaM/CaMK Ⅱ signaling pathway in liver regeneration after rat partial hepatectomy. It can make sense for liver regeneration after partial hepatectomy.