Tyrosinase small interfering RNA effectively suppresses tyrosinase gene expression in vitro and in vivo.

Tyrosinase is a bifunctional enzyme which oxidizes the initial step of melanin biosynthesis, that is, conversion of tyrosine to dopa and subsequently dopa to dopaquinone. It is a glycosylated protein and a major regulator of melanogenesis. To date, many approaches have been tried to regulate tyrosinase activity and melanin content.

[siRNAs targetting sphingomyelin phosphodiesterase 1 protect mouse oocytes from apoptosis].

Objective: To investigate the potential of siRNAs targeting sphingomyelin phosphodiesterase 1 (SMPD1) in protecting the oocytes from apoptosis, and explore new approaches to female fertility preservation.

Methods: Chemically synthesized siRNA targeting SMPD1 were introduced into mouse oocytes retrieved by hyperstimulation, and the cell apoptosis was analyzed by comic assay 48 and 72 h later.

Results: In the oocytes without any siRNA injection, oocyte DNA damage occurred after 24 h, and large amount of DNA fragments migrated from the cells 48 h later.

Phenotypic changes of human cells in human-rat liver during partial hepatectomy-induced regeneration.

Aim: To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera (HRC) generated by in utero transplantation of human cells during partial hepatectomy (PHx)-induced liver regeneration.

Methods: Human hepatic parenchymal and stromal components and phenotypic changes of human cells during liver regeneration were examined by flow cytometry, in situ hybridization and immunohistochemistry.

Results: ISH analysis demonstrated human Alu-positive cells in hepatic parenchyma and stroma of recipient liver.

A study of using tissue-engineered skin reconstructed by candidate epidermal stem cells to cover the nude mice with full-thickness skin defect.

Objective: To study and explore the feasibility of using candidate epidermal stem cells with reconstruct tissue-engineered skin for a skin defect.

Methods: After the candidate epidermal stem cells were selected directly by rapid adhesion to type IV collagen within 10min from keratinocytes isolated from foreskin epidermis, the TES was constructed by seeding large-scale cultured candidate epidermal stem cells onto a fibroblast-containing dermal substrate, then grafted onto athymic immunodeficient mice with full-thickness skin defects. All specimens were harvested after 1 week, 2 weeks and 4 weeks of transplantation to evaluate by gross, histological, transmission electron microscopic and immunohistochemical techniques its potential to reconstitute a full-thickness skin defect.

Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo.

Aim: To accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed.

Methods: Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients.

Results: Of 29 live-born recipients, 19 had the presence of human CD45(+) cells in peripheral blood (PB) detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues (i.

High expression of hepatitis B virus based vector with reporter gene in hepatitis B virus infection system.

Aim: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector.

Methods: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009.

[Mesenchymal stem cells do not differentiate into "quasi-sperm"].

Objective: To determine whether the surviving mesenchymal stem cells (MSCs) in the testis after transplantation can differentiate into quasi-sperm.

Methods: (1) Making an animal model with sterilized testes. Forty 4-week old white male BASB/C mice were used to establish an animal model with sterilized testes and divided randomly into an experimental and a control group.

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras.

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat.

[Making of the animal model with sterilized testes].

Objective: To explore the methods of making an animal model with sterilized testes.

Methods: (1) X-ray local irradiation. Seventy 8-10-week-old male mice were equally divided into 6 experiment groups and a control group.

Rescue of the albino phenotype by introducing a functional tyrosinase minigene into Kunming albino mice.

Aim: To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the efficiency of gene transfer.

Methods: A mouse tyrosinase minigene, i.e.

Overexpression of hepatitis B virus-binding protein, squamous cell carcinoma antigen 1, extends retention of hepatitis B virus in mouse liver.

How receptors mediate the entry of hepatitis B virus (HBV) into the target liver cells is poorly understood. Recently, human squamous cell carcinoma antigen 1 (SCCA1) has been found to mediate binding and internalization of HBV to liver-derived cell lines in vitro. In this report, we investigate if SCCA1 is able to function as an HBV receptor and mediate HBV entry into mouse liver.

[Transplantation of mouse bone marrow mesenchymal stem cells into the xenogeneic testis].

Objective: To study transplantation of mouse bone marrow mesenchymal stem cells (MSCs) into the xenogeneic testis.

Methods: (1) The tibias and femurs were dissected from 5-6-week-old mice. The marrow in the tibias and femurs was flushed out with medium.

[Isolation and culture of mouse marrow mesenchymal stem cells (MSCs) and biological characteristics of MSCs cultured in conditions for spermatogonia in vitro].

Objective: To isolate, culture and purify mouse bone marrow mesenchymal stem cells (MSCs) and observe the main biological characteristics of MSCs cultured in conditions for spermatogonia in vitro.

Methods: The tibias and femurs were dissected from 5 - 6-week old mice and the marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured, purified in vitro by Percoll density gradient centrifugation combined with adherent method and identified by dynamic observation of stem cell characteristics by transmission electron microscope, HE staining, and immunohistochemical detection of cell markers.

Generation of the regulatory protein rtTA transgenic mice.

Aim: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome "immune tolerance" formed during the embryonic development and "immune escape" against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated.

Methods: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, i.

Malignant transformation of the cultured human hepatocytes induced by hepatitis C virus core protein.

Background/aim: Hepatitis C virus core protein (HCV-C) has been known to play an important role in hepatocarcinogenesis. But, up to now there is no certain evidence in pathomorphology directly supporting this standpoint. In this study, a human hepatocytes model expressing HCV-C was established for investigating the influence of HCV-C on hepatocytes biological properties.

Temporal control of Cre recombinase-mediated in vitro DNA recombination by Tet-on gene expression system.

Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering.

Temporal and tight hepatitis C virus gene activation in cultured human hepatoma cells mediated by a cell-permeable Cre recombinase.

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/lox P switching expression system, we plan to develop efficient conditional transgene activation of hepatitis C virus core protein (HCV-C) cDNA (nucleotide 342-914) in the transgenic mice to overcome "immune tolerance" formed during the embryonic period and "immune escape" against hepatitis virus antigen in our project. To use this system in vivo, the dormant transgenic construct, i.

[A pilot study on transdifferentiation of skin stem cell in reconstructing corneal epithelium].

Objective: To observe the differentiation and development of skin stem cells on corneal stroma and to discuss the possibility of reconstructing corneal epithelium with skin stem cells.

Methods: Pieces of human and rabbit skin were obtained during operation. Rabbit eye balls were taken, and pieces of corneal stroma without epithelium were prepared.