The Effect And Mechanism Of Tg737on Differentiation Of Fetal Liver Stem Cells

Fetal liver stem cell (FLSCs) can differentiate into hepatocyte and biliary epithelialcell, this feature would build a broader platform for basic research and clinicalapplication. The visualize and practice of stem therapy for various liver diseases coincidewith the regenerative medicine’ s idea, its future is bright and ambitious. At the sametime, immense amounts of research indicated there is a subtle connection between stemcell and cancer stem cell, abnormal differentiation of stem cells might lead to theinitiation of cancer. Therefore, both positive and negative aspects of stem cell biologicalproperty exhibits the importance and necessity of our study, mainly to investigate thedifferentiation course of FLSCs, to explore which key molecule, protein and/or signalingpathway play a critical role and potential regulate mechanism in the process of FLSCs’differentiation.ObjectiveThe normal differentiation of FLSCs as an entry point prompts us to investigate, inthis context, a tentative exploration concerning the effect and mechanism of Tg737in thedifferentiation process is carrying out. Research contents are split into two aspects:1. the expression and role of Tg737gene in the course of FLSCs’ differentiation.2. the potentialmechanism on Tg737gene to regulate FLSCs’ differentiation.Method1. A convenient and efficient way that is called three-step isolation method which isinvented by our research group was used to collect FLSCs. FLSCs was induced via HGF(20ng/ml) for one week, the1,3,5and7day were defined as observation point infollow-up experiment. The successive mRNA expression of ALB and AFP in normalFLSCs was tested by PCR. Using Western blot to detect protein variation of Tg737atabove mentioned four point. FLSCs was transfected by lentivirus Tg737-shRNA, dividingFLSCs into experiment and control group, using PCR and Western blot to test silenceeffect. Using Flowcytometrty to detect the expression of CD133and PCR to continuousmonitor mRNA change of ALB and AFP in two groups. At the7day, the FLSCs’ultrastructure of two groups was observed via transmission electron microscope (TEM).2. The mRNA and protein level of HNF4α in the process of FLSCs’ normaldifferentiation was successive detected by PCR and Western blot. At the7day, using PCRand Western blot to test the level of HNF4α’s mRNA and protein, immunofluorescencetechnique to probe β-catenin’ expression and Western blot to detecte the proteinexpression of Snail respectively in two groups.Results1. FLSCs can be enriched efficiently by what is called three-step isolation methodwhich is invented by our research group. The mRNAlevel ofAFP was decreased little bylittle at the1,3,5and7day under inducing FLSCs by HGF (20ng/ml), the contrarymRNAexpression was emerged inALB. The protein expression of Tg737was increasedby degrees at the induced differentiate course. The tool of lentivirus-shRNAcandown-regulate Tg737gene’s mRNAand protein resultfully in FLSCs. In the process ofHGF-induction FLSCs, The expression of CD133in experiment group manifested moreslowly downtrend compared with the control group during induced differentiation course,except the first day (p<0.05). the mRNAlevel ofAFP andALB had no obvious change inexperimental group (p>0.05). TEM demonstrated that experiment group’FLSCs presented morphological characteristics of low differentiation status, and control groupexhibited morphological features of early differentiation status which was similar to thoseof hepatocytes.2. Continuous monitoring comes from PCR and Western blot showed the level ofHNF4α mRNA and protein increased gradually in FLSCs at the1,3,5and7day via HGF(20ng/ml)-stimulated (p<0.05). The mRNA and protein expression of HNF4α inexperimental group showed down-regulation significantly (p<0.05), meanwhile,intranuclear β-catenin presented more stronger immunoreactivity compared with thecontrol group, the protein expression of Snail was up-regulated in Tg737-inhibition group.Conclusion1. FLSCs can be extracted and purifed effectively via procedures including PDGC,DTDA and PCGC as described in our previously study. FLSCs can be induced to divideinto normal hepatocytes by HGF (20ng/ml).2. Tg737gene participates in the differentiation process of FLSCs, inhibitedexpression of Tg737in FLSCs leads to differentiation arrested.3. HNF4α also takes part in the normal differentiation of FLSCs. Deletion of Tg737gene resulted in down-regulated expression of HNF4α and excessive activation ofWnt/β-catenin signal pathway, at the same time, the process of EMT in FLSCs may bepromoted, such changes caused abnormal differentiation of FLSCs.