Effects of lentiviral vector­mediated shRNA silencing of TGFß1 on the expression of Col1a1 in rat hepatic stellate cells.

The present study aimed to construct a lentiviral RNA interference (RNAi) vector targeting the transforming growth factor ß1 (TGFß1) gene of rats, in order to examine its effect on silencing of the TGFß1 gene and on the expression of collagen type 1 α1 (Col1a1) in HSC­T6 rat hepatic stellate cells. Three RNAi sites of the TGFß1 gene were selected according to its CDs sequence. Three pairs of small interfering RNA (siRNA) of these RNAi sites were synthesized and then transfected into HSC­T6 cells, respectively, to confirm the optimal siRNA sequence via reverse transcription­polymerase chain reaction analysis. Subsequently, shRNA targeting the sequence of the optimal siRNA was designed, synthesized and annealed to form a double­stranded structure. The annealed oligonucleotide fragment was cloned into pGreenPuro plasmids to establish the pGreenPuro/TGFß1 shRNA lentiviral vector, which was then transfected into 293T cells, following identification by restriction enzyme digestion and sequencing for the production of lentiviral particles exhibiting high reactivity. These particles were used to infect HSC­T6 cells, following which the expression of GFP in the transfected cells was observed under an inverted microscope. The effects on TGFß1 gene silencing and the expression levels of Colla1 were detected at the mRNA and protein levels. The results provided confirmation of the optimal siRNA sequence. Enzyme digestion and sequencing verified successful construction of the pGreenPuro/TGFß1 shRNA lentiviral vector. This lentiviral vector effectively silenced the TGFß1 gene in the HSC­T6 cells, and inhibited the expression of Col1a1 at the mRNA and protein levels. Taken together, the lentiviral RNAi vector targeting the TGFß1 gene of rats was successfully constructed, which effectively silenced the TGFß1 gene of the HSC­T6 cells and inhibited the expression of Col1a1.

Cytokine-induced killer cells combined with dendritic cells inhibited liver cancer cells.

OBJECTIVES: To investigate the prognosis of advanced liver cancer patients treated with CIK-DCs and the mechanism of apoptosis of HEPG 2 cells. METHODS: 67 patients were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were separated, of which adherent PBMCs used granulocyte 2 macrophage colony2 stimulating factor (GM2CSF), tumor necrosis factor 2α (TNF2α), and interleukin 24 (IL24) to induce DCs, which were sensitized with antigen of autologous or exogenous cancer cells to obtain Ag-DCs; suspended PBMCs used interferon 2γ (IFN2γ), IL-2, and CD 3 monoclonal antibody (CD3mAb) respectively, to induce CIK cells. DCs and CIK cells were cultured together. Flow cytometry was used to detect the phenotypes of DCs and CIK cells, and the blood retransfused into patients. Western blot and flow cytometer were used to analyze the growth cycle of HepG 2 cells and the expression of BAX and PCNA. RESULTS: No patients underwent complete remission, 5 obtained partial remission and 29 had stable disease. Of the 31 patients whose lesions could not be evaluated, 17 received effective treatment, showing that the immune response was enhanced. In vitro laboratory experiments revealed that DC-CIK cells markedly affected the growth cycle of HepG 2 cells. Analysis showed that DC-CIK cells enhanced the gene expression of BAX and inhibited the activity of PCNA. CONCLUSIONS: Co-cultured DCs and CIK cells inhibit the proliferation and migration of liver cancer cells by down-regulating PCNA and up-regulating BAX. This approach may be an effective method to treat advanced liver cancer.

[Exogenous gene expression in vitro and in vivo in bone marrow mesenchymal stem cells modified by hPDGF-A and hBD(2)].

The objective of this study was to investigate the expression of exogenous hPDGF-A and hBD(2) in gene-modified bone marrow mesenchymal stem cells (BM-MSCs) in vitro and in vivo. Recombinant adenovirus vector expressing hPDGF-A/hBD(2) genes was constructed and packaged into virion. Primary isolated and cultured BM-MSCs were transfected by using hPDGF-A hBD(2), then the expressions of exogenous hPDGF-A/hBD(2) were detected by immunocytochemical staining in vitro. The conditioned medium (serum-free cultured supernatant of BM-MSCs transfected with recombinant adenovirus) collected from gene-modified BM-MSCs was applied to scratch wound on monolayer cells of multipotential cell line 10T1/2 in order to confirm the stimulative effect of hPDGF-A on cell migration. Gene-modified BM-MSCs were topically transplanted on wound of rats with radiation and skin excision combined injury. The distribution of BM-MSCs and expression of hPDGF-A/hBD(2) on the wound was observed by fluorescent microscopy and immunohistochemical staining respectively. The results indicated that the rat BM-MSCs transfected with recombinant adenovirus could express the EGFP in vitro. The immunofluorescent cytochemistry assay showed that the gene-modified BM-MSCs expressed the hPDGF-A and hBD(2). The scratch test confirmed that the percentage of healing area of wound in cultured supernatant group of gene-modified BM-MSCs was significant higher than that in control group on 8, 12, 24 and 48 hours (p < 0.05). The fluorescence microscopy of exogenous gene-modified BM-MSCs transplanted on wound revealed that the gene-modified BM-MSCs could higher express exogenous genes of EGFP at least within 2 weeks. The immunohistochemistry staining of wound indicated that the expression of exogenous genes began from day 3, reached to peak on day 7, and still visible on day 21 even though the expression became weak because of the possible dilution of the exogenous genes during cell division. It is concluded that efficient expression of exogenous hPDGF-A/hBD(2) in gene-modified BM-MSCs are demonstrated both in vitro and in vivo, which suggests that the molecular mechanism underlying chronic wound-healing accelerated by the strategy combining cell therapy with gene therapy.

Effects of peritoneal lavage fluid from radiation or/and burn injured rats on the growth of hematopoietic progenitor cells.

PURPOSE: To evaluate the effects of peritoneal lavage fluids from radiation injury, burn injury and combined radiation-burn injury on the growth of hematopoietic progenitor cells (HPC). MATERIALS AND METHODS: Rats were divided into four groups: A radiation group (RG), a burn group (BG), a combined radiation-burn group (CRBG) and normal control group (NG). RG and CRBG rats were irradiated with 12 Gy, and burns of 30% total body surface area were generated in group BG and group CRBG. Peritoneal lavage fluids were collected and tested for their effects on the growth of erythrocyte progenitor cells or granulocyte-macrophage progenitor cells of BALB/c mice in vitro. RESULTS: The numbers of colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) formed after treatment with lavage fluids from BG or CRBG were significantly higher than those from NG. However, fewer CFU-E, BFU-E or CFU-GM colonies were found after treatment with lavage fluid from the RG. In lavage fluid from BG and CRBG, the concentration of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNFalpha) was increased in comparison to NG and RG. Treatment with these cytokines had similar promoting effects on the growth of hematopoietic colonies and neutralizing antibodies inhibited these effects significantly. CONCLUSIONS: Burns increase the responsiveness of the system and help the proliferation of hematipoietic progenitor cells, while radiation decreases all these responses relative to both the controls and the burn plus radiation group.