Prokaryotic Expression And Purification Of CKIα In Rats, Construction Of Carrying Rats CKIα Lentiviral Vector And Experimental Study On BMSCs In Transfected Rats

Liver disease poses a serious threat to human health. Each year, according to the statistics,about1.4million people worldwide die of advanced terminal liver failure end-stage liver failure caused by chronic liver disease including hepatitis, infections and tumors. Orthotopic liver transplantation (Orthotopic Liver Transplantation, OLT),as the most important treatment for liver failure at present, is difficult to carry out extensively due to the shortage of liver source and high costs, which makes a limited number of people benefit from it. As a complementary or alternative therapies for liver transplantation, growing concern has been attached to the bioartificial liver (Bioartificial Liver, BAL) and liver cell transplantation (Liver cell Transplantation, LCT) in recent years. However,lack of cell sources has been a major bottleneck in the development and application of both BAL and LCT. The stem cell technology, which has been developed since1990s, becomes a hot research in the field of life sciences. Stem cell transplantation, as a newly-emerging means of cells therapy, provides a new thought and method for solving the source of seed cells and the treatment of liver disease.Bone Marrow Mesenchymal Stem Cells (BMSCs), early undifferentiated cells with self replication, differentiation potential and high plasticity, belong to a kind of adult stem cells. BMSCs promise to be ideal seed cells of tissue engineering and cell therapy because they can cross blastoderm and transdifferentiate to multiple tissue cells like hepatocyte-like and possess the good qualities of being available conveniently, in vitro culture maturation and autotransplantation. How to induce transdifferentiation of BMSCs into hepatocyte-like is a key to stem cell transplantation in treatment of hepatopathy and has not been settled satisfactorily due to its differentiation mechanism still not being clear, which is far from the desired effect in clinical application. Therefore it is of great significance to explore molecular mechanism of differentiation of BMSCs into hepatocyte-like.Many experiments has confirmed that BMSCs can differentiate into hepatocyte-like but its mechanism still remains controversial. The main view of point at present are as follow:①transdifferentiation theory:BMSCs are multipotential and capable of transdifferentiation to hepatocyte-like.②cytomixis theory:BMSCs first mix with The host liver cells and then further differentiate into hepatocyte-like. Although definite regulation mechanism of transdifferentiation is still not clear, the majority of scholars are supportive of it since cell fusion is not found in many experiments of stem cell differentiation.It is the microenvironment on which stem cells rely for existence, so many researchers regard it as the point of contact of directional differentiation of stem cells. The local microenvironment of cells includes various cytokines, hormones, matrix cell and extracellular matrix around cells. Of all, cytokines is of vital importance. Expression form, concentration and action order of the various factor in the microenvironment are the important factors which influence BMSCs differentiation. Stem cells are influenced by different composition and structures of microenvironment in different stages, different location and different circumstance. Under normal circumstances, microenvironment is used to protect stem cells from clearing away, keep their undifferentiated state, and restrain their excessive proliferation. When the body suffers damage, microenvironment change correspondingly to selectively activate and restrain relevant transcription factors through certain signal channel deliver and start related gene, accordingly particular regulation of transcriptional and translational level makes stem cells differentiating to particular adult cells.Based on the above point of view, we put forward certain biological factors Intrahepatic microenvironment, different from physiological state, can stimulate differentiation of BMSCs into hepatocyte-like. To explore which biological factors paly an role of inducing, our research group compared rats with hepatic failure with healthy rats by proteomics technology, selected27protein loci which has significant differences. We use bioinformatics analysis and found up-regulating casein kinase Iα and T box of transcription factors, and down-regulating tyrosine protein kinase are closely related to cell proliferation.Casein kinase lα (CKIα) is a serine or serine-threonine kinase with special physicochemical properties, widely existing and distributing in eukaryotes, mainly distributing in cytoplasm, cell membrane and so on. CKIα possesses the feature of constitutive activity, that is, separated CKIα or prokaryotic expression CKIα have activity. Phosphorylated loci among CKIα phosphorylated protein substrates can be used to be other protein kinase substrate phosphorylated and participate in protein cascade phosphorylation process, which then join multiple cell regulation process including gene expression, cell proliferation and differentiation through signal channel. Study have proved that CKIα mainly participates in signal channel of Wnt and Hh which are closely related to differentiation and proliferation of stem cells.CKIα expression is significantly increased in the microenvironment of liver failure.But where are they coming from? It remains unclear whether it comes purely from the release of the damaged hepatocyte or the secretion of stem cells stimulated by microenvironment. Is the up-regulated CKIa which can promote hepatocyte-like differentiation of BMSCs caused by BMSCs stimulated directly by exogenous CKIa, or by BMSCs overexpression CKIa induced by certain factors in the microenvironment and then the endogenous expression CKIa promoting hepatocyte-like differentiation of BMSCs? That is what we want to research. Therefore,in the first part of the study, we intend to efficiently and steadily express CKIa from Escherichia coli by employing molecular biological technique and prokaryotic expression system and purify the recombinant CKIa through Ni-chelating affinity chromatography efficiently, which prepares for the next experimental study on CKIa co-cultured with BMSCs and lays the groundwork for further protein biological functional identification and study on action of differentiation of CKIa to hepatocyte-like. In the second part of the study,we plan to construct the lentiviral vectors which can be induced to express genes of CKIa by the Gateway technology. After that we want to establish a stable model of BMSCs overexpress CKIa by transfected vectors,which will lay the foundation for the further experiments of differentiation of BMSCs into hepatocytes in vitro and in vivo.Chapter I prokaryotic expression and purification of CKIa in ratsObjective:To construct CKIa expression plasmid in rats, prepare and purify recombinant CKIa by E. coli prokaryotic expression system and get recombinant CKIα with heavy concentration and high purity.Methods:RNA extract kit separated and purified total RNA of rat liver cells. The first chain of cDNA was compounded by using RNA as template and random hexamer nucleotide as primer under action of M-MLV reverse transcriptase. According to code sequence of rat CKIa reported by GenBank, two pairs of primers of rat CKIa was designed to be amplified, restriction enzyme cutting sites (Ndel and BamHI) were added in the5-prime end of the inside of the upstream and downstream primers, respectively. TaqDNA polymerase was used to do nested PCR amplification CKIa gene by using cDNA as template. PCR amplified products were separated by electrophoresis and purified by low-melting-point agarose gel, and then inserted into Prokaryotic expression plasmid pET-16b to construct recombinant plasmid pET-16b-CKIa. After it was digested and got DNA sequencing identification, pET-16b-CKIα was transformed into escherichia coli Stbl3competence bacteria. Then engineering bacteria (1:100) was inoculated in LB culture medium that contained glucose and glycerin were cultured at37℃until OD value was about0.5-1.0. Three hours later of cultivation, collect bacteria by centrifuge, wash and separated inclusion body at1200r/min after ultrasonic decomposition, wash sediment three times with0.5%Triton and purify inclusion body which is then dissolved’by8M urea. At last, gel filtration was used to purify interest protein and identification of recombinant CKIα samples were analyzed by Western blotting.Result:1. Nucleotide sequence determination and NdeI/BamHI double digests identification suggest that the recombinant plasmid pET-16b-CKIa was managed to be constructed;2.38kDa protein bands denseficated obviously were found between30kDa and40kDa and the corresponding exposure bands in film were consistent with the expected size after recombinant CKIa through affinity and purification of Ni-NTA was detected by Western blotting.Conclusion:1. pET-16b-CKIα prokaryotic expression vector, which expressed recombinant protein of CKIα after being transformed into escherichia coli, was constructed successfully;2. Preliminary researches optimized expression condition, extraction steps of inclusion body and preliminary purification technology of recombinant CKIa in rats;3. Clone rat CKIa gene successfully, and sequence analysis coincided with literature reports;4. Purification of recombinant CKIa laid a certain foundation for further Protein biological functional identification and study on effect of CK la on differentiation of BMSCs to hepatocyte-like. Chapter Ⅱ Construction of rats CKIa lentiviral vector and experimental study on BMSCs in transfected ratsObjective:To construct lentiviral vector of inducible expression CKIa gene, transfect Human embryonic kidney epithelium cell lines293FT cells got the corresponding virion, infect and obtain BMSCs in rats with stable and efficient expression of CKIa under inducing condition of doxycycline, lay the foundation for further rat BMSCs transplant research of inducible expression CKIa.Methods:pcDNA-CKIa as template, polymerase chain reaction was employed to amplify overall sequence of CKIa at back and front end with attB recombinant loci. Gateway vector technique was used to clone amplified product to pDown vector by BP reaction. After correct sequencing identification, LR recombinant enzyme was used to catalyze recombinant reaction of pDown-CKIa, pUP-EF1A and PLV.Des3d. p/neo to construct lentiviral vector expression plasmid pLV.Des3d.P/neo-EF1A-CKIa-IRES/eGFP which was then mixed with packaging plasmid (pLV/helper-SL3, pLV/helper-SL4、pLV/helper-SL5). Together with the previous plasmids, lipidosome was used to transfect293FT cells, package lentiviral particle carrying with CKIa gene, infect rat BMSCs. Under inducing condition of doxcycline, RT-PCR and Western blotting was used to detected expression condition of recombinant CKIa gene. Results:Digestion, polymerase chain reaction and sequence identification proved lentiviral vector of inducible CKIa eukaryotic expression was construct successfully; pLV.Des3d.P/neo-EF1A-CKIa-IRES/eGFP and packaging plasmid co-transfected packaging cell293FT, which made lentiviral particle carrying with CKIa gene achieved successfully. Viral drop degree generated by package was5×107TU/ml in CKIa. under inducing condition of doxycycline, infection rate detected by fluorescent microscope is up to above90%.RT-PCR were used to detected expression condition of recombinant CKIa gene and verified gene level expression. Transfected group shown positive while untransfected group shown negative. Western blotting was used to detected expression condition of CKIa and verified transfected cells and supernatant expression are positive while untransfected cells expression are all negative.Conclusion:Lentiviral vector of inducible CKIα expression was construct successfully and condensed lentiviral-CKIa liquid was achieved; Achieving BMSCs in rats with stable and efficient expression of CKIa under inducing condition of doxycycline laid the foundation for further BMSCs transplant research of inducible expression CKIa.