The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo.

Mesenchymal stem cells (MSCs) play an important role in the development and growth of tumor cells. The purpose of this study is to confirm the effect of MSCs on tumor cell growth in vitro and in vivo and to elucidate the mechanism. MSCs were isolated from mouse bone marrow and cocultured with murine hepatoma H22, lymphoma (YAC-1 and EL-4) and rat insulinoma INS-1 cell lines. The growth inhibitory effect of MSCs on tumor cells was tested through MTT and 3H-TdR incorporation assay. The apoptosis induction effect of MSCs on tumor cells was assessed with flow cytometry (FCM) and RT-PCR assay. MSCs were inoculated into BALB/c mice alone or coinoculated with ascitogenous hepatoma cells intraperitonealy, respectively. The tumor growth inhibition of MSCs was investigaed through the incidence and volume of ascites formation, and the immunosuppression effect was studied with splenocyte response to ConA stimulation test and T cell subsets analysis (FCM). The results showed that MSCs exhibited a number-dependent growth inhibitory effect on murine tumor cell lines in vitro and inhibited the growth of ascitogenous hepatoma cells in vivo without host immunosuppression. MSCs could upregulate tumor cells mRNA expression of cell cycle negative regulator p21 and apoptosis associated protease caspase 3. The findings of this experimental study demonstrated that MSCs had potential inhibitory effects on tumor cell growth in vitro and in vivo without host immunosuppression, by inducing apoptotic cell death and G(0)/G(1) phase arrest of cancer cells.

[Construction of a cDNA library from liver tissue of rhesus monkey, Macaca mulatta].

OBJECTIVE: To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a cDNA expression library from liver tissue of a healthy rhesus monkey. METHODS: With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), the first-strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand cDNA, which was subsequently digested by Xho I restriction enzyme and fractionated with CHROMA APIN-400 column. The fractionated cDNA fragments to be longer than 0. 5 kb were ligated into lambda ZAP express vector to form the phagemid cDNA recombinants, which were further packaged into the lambda ZAP cDNA library according to the standard protocol with phage lambda Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF'. Then, the library titre, recombinant rate and length of inserted cDNA were measured, respectively. RESULTS: The capacity of the primary stand or unamplified library was 1. 2X 10(6) pfu. The titers of the unamplified library or the amplified library was 1.1 X 10(6) mixture, pfu/mL or 7. 7 X 10(9) pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%, and the average lengths of the inserts were 2.0 kb and 2. 3 kb, respectively. CONCLUSION: An excellent cDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.

Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line.

BACKGROUND: Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line. METHODS: A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme. RESULTS: Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences. CONCLUSION: These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.