Influence Of Tmub1Gene Silencing On Rats’ Hepatic Cell Proliferation Cycle After Partial Hepatectomy

Background：Partial hepatectomy （PH） is the most efficient way to cure variousinnocent and malignant hepatic diseases at present. But, it is likely to cause fateful liverfailure after extended PH, which is a worldwide difficult in medical treatment. Althoughliver transplantation is an effective therapy method, liver transplantation is limited in itswide development due to seriously insufficient donors. The hepatic function is unable to becompletely replaced by the existed bioartificial liver technology. In addition, the therapeuticeffect is also restricted due to limited proliferation of hepatic cells transplanted to humanbodies. But, different from other organs, liver has a powerful potential regenerationcapacity. At present, it is widely believed that remnant liver regeneration after PH isrealized via the remnant liver cells’ replication and proliferation. Currently, less study hasbeen made on hepatic cells’ regulation mechanism, and it is recorded in fewer literaturesthat TGF-β may be reflected in negative regulation of hepatocellular proliferation.Therefore, it has a extremely important practical significance to explore regenerationmolecules mechanism of hepatic cells, to lay a theoretical foundation for promoting hepaticregeneration and preventing liver failure clinically.It is shown from the research topic supported by NSFC that: Tmub1protein plays animportant self regulation role for the proliferation process during the proliferation cycle ofhepatic cells. Tmub1was reported in2005firstly that Tmub1protein contains245aminoacids, located in amino terminal and a UBL（121-175aa） where reaction sites with UCH, E2and CUE. At present, the results of study on Tmub1gene and its protein product include:Tmub1protein is a kind of shuttling protein. With excessive expression of Tmub1, rats’H-35hepatoma cells’ proliferation can be obviously restrained. After Tmub1silencing,centrosome will becomes abnormal, apocyte will appear, and abnormal spindle will form. Itis approved by the research results that Tmub1gene and its protein product are complex infunctions, especially that they may play an important regulation role in the liver regeneration cells’ proliferation cycle, but the specific regulation mechanism is not clear.At present, external hepatic cells’ level gene transfection has been reported for times,and the transfection technology has become well-developed. But, it is different in researchmethods on how to transfect the external target gene into the liver. In some researches, genetransfection is realized via rat’s caudal vein injection, but lots of virus plasmids shall beinjected during test, the rat may have an excessive load than physiologic amount, and evenmay die, and also the transfection efficiency is low; a general practice is that the genetransfection is realized via rat’s portal vein injection, in such way, virus injection will bereduced and also the transfection is obviously higher. But, the portal vein injection positionis very deep, it is likely to cause rat’s death due to bleeding, and therefore the experimentwill fail. What’s more, through portal vein injection, it is likely to cause the change ofblood flow entering liver and then the change of hepatic sinus pressure, which may result inthat hepatic sinus cells secrete various cell factors, thereby causing the change of originalinternal liver environment and correspondingly potential experimental interference will becaused. As for the problem on how to optimize the liver gene transfection routes so as tofurther improve the liver research methods and clinical application, it has been not reporteduntil now.Objective:1. To discuss the experimental model of liver gene tranfection to rat’s body and findout a kind of gene transaction method which is easy to operate, has a small stimulation onrat’s response, and has a higher gene transfection rate.2. To observe the change of hepatic cells’ proliferation cycle process after PH and theinfluence of Tmub1on liver regeneration through Tmub1gene silencing.3. To preliminarily discuss the regulation mechanism of Tmub1protein on hepaticcells’ proliferation cycle, and analyze the possible relevant regulation protein andmechanism of action.Methods:1. Experimental study on rats’ liver Tmub1RNAi lentivirus transfection and effectevaluation1.1Experiment on rats and rats grouping. As a rat’s caecum vein is relatively large andlocates in a shallow position, it is easy to find it and the operation is simple; moreover, there are rich adipose tissues around, and caecum vein belongs to a branch of portal vein,therefore the rates are grouped, namely, caecum vein injection group, portal vein injectiongroup, caudal vein injection group, and control group, and they will be injected withreorganized Tmub1RNAi lentivirus carrying GFP.1.2Frozen and section the liver tissues on the next day of gene transfection, observeeach group’s GFP expression conditions with laser scanning confocal microscope （LSCM）,and then make a comparison on transfection rate between groups.1.3Assess the influences on portal vein pressure of injection via portal vein andcaecum vein. Arrange3rats respectively in caecum vein injection group and portal veininjection group, use Powerlab/8sp data acquisition unit（ADinstruments, AU）to test theportal vein pressure changing conditions during injection via portal vein and caecum vein,and then compare the influence on portal vein pressure under these two kinds of injectionmethods.1.4Inspect the influence on rats’ liver functions and organism stimulation responsesunder three kinds of injection methods. On the second day of injection to rats, take5mlnon-anticoagulative portal vein blood respectively from the rats, and obtain blood serumafter the portal vein blood is centrifuged, and detect each sample for alanineaminotransferase（ALT）, aspartate transaminase（AST）, Tumor Necrosis Factor-a（TNF-a）andInterleukin-6（IL-6）values for at least three times, and then statistics analysis.2. Research on the influence of Tmub1silencing on liver regeneration cells’proliferation period after PH2.1Rats experiment and grouping. After a successful internal liver transfection, copyclassical70%rat PH model, extract the primary hepatic cells and tissues as samples, andresearch the influences of Tmub1silencing on liver regeneration cells’ proliferation periodafter PH. Arrange36rats in three groups, namely Tmub1RNAi experimental group, lentivirus empty vector group, and control group, and respectively select3rats at4time phasepoints like0h,2h,12h and24h after PH in each group.2.2Respectively detect the mRNA level and protein expression conditions of Tmub1gene and securing gene in the liver cell samples extracted with RT-PCR method andWestern Blot method. Among which, the detection on Tmub1reflects the RNAi lenti virusinterference effect and Tmub1silencing effect, while the detection on securin reflects the influence of Tmub1silencing on gene level and protein level.2.3Detect the cell proliferation conditions of each group with MTT experiment.Collect each group’s cells extracted after HP and place them in the nutrient mediumcontaining serum; dilute cells to25×103/ml, then add MTT agent, incubate the culture plateunder37℃wet environment for3h, detect the OD value of each pore with microplatereader, and draw the cell proliferation curve.2.4Observe the cell proliferation conditions of each experimental group with FCM（flow cytometry）. Fix the cells extracted from each experimental group. Firstly fix the cellswith70%alcohol, add RNA enzyme and P1, then detect the cells with FCM half an hourlater, and immediately analyze the data results.2.5Detect the interaction between Tmub1protein and securin with IP and proteinmass spectrum analysis method. Add proper protein extracted from cell IP splitting buffersolution to cells, add10-50μl anti-securing to cell splitting solution and slowly shake thesolution, collecte securin/G-beads and then carry out Western blotting electrophoreticanalysis and protein mass spectrum analysis.Results:1. Experimental study on rats’ liver Tmub1RNAi lentivirus in-vivo transfection andeffect detection1.1Conditions on injection of slow virus to rat’s liver in vivo. Through statistics on theaverage injection time of three groups of experimental rats, it is discovered that theinjection via the portal vein is the most complicated and time-consuming, while theinjection via caudal vein is most convenient, and the average time of injection via caecumvein is about8-17minutes. Meanwhile, through the comparison on success rate of threegroups of experimental rats, it is discovered that the injection operation via caecum veinand caudal vein are both successful in all rats, but the injection via the portal vein results inthree rats’ death due to hemorrhage inside abdominal cavity. It is found that the blood canbe stopped after being pressed for ten seconds after injection due to rich adipose tissuesaround the caecum vein, so the success rate in operation of injection via caecum vein is100%.1.2Observing the GPF expression of frozen liver sections with laser scanningmicroscope. Stronger green fluorescence can be obviously observed in both caecum vein injection group and portal vein injection group; these two groups have basically consistenttransfection effects; and the green fluorescence is expressed on hepatic cell cords. Butscattered fluorescence is only observed in caudal vein injection group, with a lowtransfection rate. No green fluorescence is discovered in rats’ liver sections of the controlgroup.1.3In the experiment on testing portal vein pressure, the pressure of portal vein isobviously increased under injection via portal vein, while under injection via caecum veinthere is less influence on portal vein and only instant fluctuation in portal vein pressure, butthe pressure returns to normal level quickly.1.4ELISA results of serum transaminase, IL-6and TNF-a. The values of serumtransaminase（ALT182.0±29.9IU/L, AST114.4±17.5IU/L）, IL-6and TNF-a（IL-6244.5±48.4pg/ml, TNF-a137.4±81.8pg/ml）in caecum vein injection group are obviouslylower than those of portal vein injection group and caudal vein injection group. Thedifference has statistics significance（n=20，P <0.001）, which indicates that caecum veininjection clearly reduces the influence on liver function and emergency stimulus responseof organism.2. Research on the influences of Tmub1silencing on live regeneration cells’proliferation cycle after PH2.1Tmub1RNAi interference effect reflected that Tmub1mRNA and proteinexpression in experiment group are both restrained effectively（p<0.05）. The experimentalresult is basically consistent with the laser and laser confocal results are basically the same.2.2It is shown from MTT experimental result that in Tmub1RNAi experiment group,the proliferation rate of hepatic cells6h-24h after PH has an obvious increase. At the sametime, it is discovered from flow cytometry detection that the G₂/M period of hepatic cellproliferation after Tmub1silencing group accounts for11.07%, which is2.68%higher thanthat of the control group, and the two groups have an obvious difference in cell numberproportion. After Tmub1silencing, hepatic cell proliferation after PH accelerates, mainlyfocusing on G₂/M period cell, which indicates that Tmub1protein decreases theproliferation rate of hepatic cells ofG₂/M period.2.3RT-PCR and Western Blot found that there is no influence on securin mRNA afterTmub1silencing, but the securin protein expression in experiment group cells of G₂/M period clearly decreases. This indicates that Tmub1has no obvious influence on genetranscription level of securin, but mainly acts on protein level, which further proves thatthere is a certain relationship between Tmub1protein and securin degradation.2.4Immunoprecipitation （IP） results show that Tmub1protein and securin can bindwith each other in hepatic cells. At the same time, it is further indicated from protein massspectra analysis that Tmub1protein and securin can combine with each other in hepaticcells and also interaction occurs between two proteins.Conclusion:1. Carry out intrahepatic gene transfection via rat’s caecum vein injection. Under thiskind of injection method, there is a small emergency stimulation on experimental animals;operation is relatively simple, and also better transfection effect can be obtained. So, it is akind of convenient and safe intrahepatic gene transfection route.2. Recombination lentivirus carrier may carry current gene interference segment andGFP probe to be used for in vivo experiments of animals, and also is able to exert its effectto interfere the target gene.3. The proliferation rate of hepatic cells in G₂/M period will be clearly acceleratedafter Tmub1silencing, which indicates that Tmub1protein can influence the proliferationprocess of liver regeneration cells and plays an important self-regulation role in liverregeneration process.4. After Tmub1silencing, cell’s securin mRNA has no changes, which shows thatTmub1has less influence on securin in gene transcription level, but mainly acts on proteinlevel. Tmub1protein affects the amount of protein of securin in hepatic proliferation cellG₂/M period in a certain way.5. Tmub1protein can combine with securin in hepatic cells and interaction occursbetween two proteins. Tmub1may inhibit the degradation process of securin in hepaticcells during G₂/M period.