Study on mitochondrial DNA diversity among 7 inbred strains of mice

Objective：To study the genetic variation of mitochondrial DNA （mtDNA） among common laboratory strains of inbred mice. Methods： The genetic polymorphism of mtDNA among 4 classical laboratory strains of inbred mice and 3 inbred strains of mice established in China was analyzed by polymerase chain reaction coupled with restriction fragment length polymorphism（PCR-RFLP） and PCR coupled with single-stranded conformation polymorphism（PCR-SSCP）. Results： With regard to the D-loop （Displacement loop, D-loop）, tRNA^ Met＋Glu＋Ile, and ND3 （NADH dehydrogenase subunit 3, ND3） gene fragments of mtDNA from these mice,no variation was revealed by PCR-RFLP at 46 restriction enzyme sites. Further analyzed by PCR-SSCP,the D-loop 5＇fragment and 3＇end fragment of mtDNA from these mice also showed no genetic variation. Conclusion： Owing to maternal mode of inheritance of mtDNA,the results indicate that these common inbred strains of mice share the same maternal lineage.

Microsatellite Genotyping for Four Expected Inbred Mouse Strains from KM Mice

Chinese Kun Ming （KM） mouse, an outbreed strain of laboratory animal, has been widely utilized in related pharmaceutical and genetic studies throughout China. However, the value of KM mice to the research community has been severely limited, partially due to the fact that well-characterized inbred strain of KM mice is not available. Several expected inbred strains from KM mice have been bred, but their genetic purity remains uncertain. In this study, four expected inbred strains of KM mice （A1, T2, N2, and N4） were chosen and their inbred degree were compared with two classical inbred mouse lines （BALB/c and C57BL/6） by analyzing the genotypes of about 30 microsatellite markers. In the four strains, A1 and N4 were homozygous at all genotyped loci, but N2 and T2 were only heterozygous at locus D15Mit16. These results indicate that the level of genetic purity/homozygousity of A1, N4, N2, and T2 inbred line is comparable to those of BALB/c and C57BL/6. This study provided the first and solid evidence for genetic purity of four expected inbred strains of KM mice. These 4 inbred mice strains should be well maintained for further characterization and utilization in genetic studies.

Experimental study on antitumor effect of arsenic trioxide in combination with cisplatin or doxorubicin on hepatocellular carcinoma

INTRODUCTIONThe main component of a traditional Chinese drug 'Pishuang'. arsenic trioxide (As2O3), has obviously selective anti-tumor effect on human hepatocellular carcinoma (HCC)in both in vitro and in vivo studies[1-5]. Due to limited effectiveness when any anti-carcinogen is used alone and obviously increased toxicity when the dose is raised, there is no exception for As2O3. Furthermore, combined chemotherapy contributes to improve therapeutic effectiveness, disperse toxicity and surmount drug-resistance,in which the combination of traditional Chinese and modern medicine has more advantages and characteristics. As a result,we made an experimental study on anti-tumor effect of As2O3in combination with cisplantin (PDD) or doxorubicin (ADM)on HCC. to investigate the possibility of AS2O3 in combination with PDD or ADM and nature of interaction between them,and to provide experimental basis for clinical application.

Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice

AIM: To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. METHODS: Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. RESULTS: On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P【0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P 【0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. CONCLUSION: Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression of Fas ligand protein of Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.

Protective effects of cyclosporine A on T-cell dependent ConA-induced liver injury in Kunming mice

INTRODUCTIONThe T-cell dependent specific liver injury in mice induced by concanavalin A(ConA) is a newly cstablished experimental liver injury model,which is considered more eligible for the study on pathophysiology of several human liver discascs,such as viral hepatitis and autommune hepatitis[1-9].T cell activation and several cytokines release had been proven to play a critical role in ConA -induced liver injury[10-19].Cyclosprine A(CsA),an effective inhibitor of activation of T lymphocytc,hes been used widely in clinical treatment,especially in autoimmune diseases and organ transplantation[20-25].In this study,we investigated the possible effect of CsA on ConA-induced liver injury in Kunning mice.

Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.METHODS： Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body （EB） culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone （DTZ）, a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells.The outgrowths were incubated in DTZ solution （final concentration, 100μg/mL） for 15 rain before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA.RESULTS： DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells （Nkx-ES cells）, which was as much as 2 wk earlier, than those in the culture of parental ES cells （wt-ES）. The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 （PDX1）, proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters.Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.CONCLUSION： Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

Fractionated radioimmunotherapy using low doses of iodine 131 labeled anti-CEA monoclonal antibody after tumor volume reduction

OBJECTIVE: To assess the efficacy of fractionated administration of radiolabeled monoclonal antibody in the treatment of metastases after tumor volume reduction surgery, various experimental therapies were studied comparatively. METHODS: A total of 200 inbred mice received tumor implantation from a murine adenocarcinoma cell line. The mice were randomly grouped to give saline, Arc-a, 131I-C50 in single or fractionated doses, cold C50, or non-specific 131I-IgG with or without surgical removal of the implanted tumor xenograft. RESULTS: In comparison to controls, animals receiving Arc-a and radioactive agents had longer survival, smaller tumor, better clinical condition, and less metastases foci. The best therapeutic response was noted after fractionated doses of 131I-C50, which showed better results in every aspect than those treated with other modalities. The favorable outcome was even more pronounced after tumor volume reduction. CONCLUSIONS: Fractionated dosing may improve the deposition of radiolabeled monoclonal antibody (McAb) and provide the best therapeutic effect on implanted tumor and metastases. Thus fractionated radioimmunotherapy (RIT) after tumor volume reduction might be a practical method with promising therapeutic results.