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.

Anti-hepatic fibrosis effects of a novel turtle shell decoction by inhibiting hepatic stellate cell proliferation and blocking TGF-ß1/Smad signaling pathway in rats.

Hepatic fibrosis (HF), a wound-healing response to a variety of chronic stimuli, is characterized by the excessive synthesis of extracellular matrix (ECM) proteins by hepatic stellate cells (HSC) and eventually the development of hepatic cirrhosis. Turtle shell pill (TSP) is a common traditional Chinese medicine used for preventing and treating HF and early hepatic cirrhosis, but its side-effects and the shortage of ingredients limit its clinical application. In addition, its mechanism of action is not clear. In the present study, we first improved the original formula of TSP to produce a novel turtle shell decoction (NTSD) with less toxicity and easier accessible materials. In a carbon tetrachloride (CCl4)-induced HF rat model, we observed that NTSD and TSP had similar effects on the improvement of liver functions in rats, including a decrease in serum alanine amino transferase (ALT) and aspartate amino transferase (AST) serum concentrations and increased albumin content in addition to a marked attenuation of CCl4-induced liver damage and fibrosis. NTSD containing rat serum inhibited rat liver stellate cell line HSC-T6 cell proliferation and induced cell apoptosis in vitro. Moreover, the NTSD treatment significantly decreased the transforming growth factor beta 1 (TGF-ß1) and Smad3 gene expression and increased inhibitory Smad7 gene expression in liver tissues of HF rats, suggesting that NTSD inhibited the ECM expression of HSC by downregulating the TGF-ß1/Smad signaling pathway. The results of our rat model study revealed that NTSD showed good in vitro and in vivo anti-HF effects via proliferation inhibition and the induction of apoptosis of HSCs and blocked the TGF-ß1/Smad signaling pathway.

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.

Topical application of hPDGF-A-modified porcine BMSC and keratinocytes loaded on acellular HAM promotes the healing of combined radiation-wound skin injury in minipigs.

PURPOSE: To evaluate the efficacy of cultured cutaneous substitute (CCS) in accelerating the healing of combined radiation-skin wound injury (CRWI) in minipigs. MATERIAL AND METHODS: Autologous porcine bone marrow-derived mesenchymal stem cells (BMSC) and skin-derived keratinocytes (SK) were infected by recombinant retrovirus expressing human (h) platelet-derived growth factor-A (hPDGF-A). CCS was constructed by loading acellular human amniotic membrane (HAM) with normal porcine BMSC and SK (BMSC-/SK-CCS) or with hPDGF-A modified counterparts (BMSC+/SK+CCS). The expression of exogenous hPDGF-A in cells and CCS was assessed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The CCS or HAM were grafted to the dorsal CRWI sites (20 Gy local irradiation plus full-thickness skin removal, diameter = 40 mm) of minipigs. Wound healing rate and pathological changes were observed. RESULTS: High levels of hPDGF-A expression were confirmed in gene-modified cells (3780 pg/ml), cultured CCS (506 pg/ml) and transplanted CCS (250 pg/ml). The transplantation of the BMSC+/SK+CCS resulted in a shorter healing time (16-18, days) (P < 0.05 vs. other groups). The healing rates ranked as BMSC+/SK+CCS > BMSC-/SK-CCS > HAM > wound control. Pathologically, there were better granulation formation and re-epithelialisation, and collagen deposition in BMSC+/SK+CCS-treated wound than those in other groups. The angiogenesis ability followed the same order as healing rate of different groups. At day 7, the area densities of vasculature in granulation tissue of group BMSC+/SK+CCS, BMSC-/SK-CCS, HAM, wound only were 15.4, 10.3, 6.0 and 5.7%, respectively, while the number densities of vasculature was 767, 691, 126 and 109 (number/mm(2)), respectively. CONCLUSIONS: Topical transplantation of hPDGF-A modified CCS may be applicable to the management of refractory wounds.

Induced endothelial differentiation of cells from a murine embryonic mesenchymal cell line C3H/10T1/2 by angiogenic factors in vitro.

A murine embryonic mesenchymal cell line C3H/10T1/2 possesses the potential to differentiate into multiple cell phenotypes and has been recognized as multipotent mesenchymal stem cells, but no in vitro model of its endothelial differentiation has been established and the effect of angiogenic factors on the differentiation is unknown. The aim of the present study was to evaluate the role of angiogenic factors in inducing endothelial differentiation of C3H/10T1/2 cells in vitro. C3H/10T1/2 cells were treated with angiogenic factors, VEGF (10 ng/mL) and bFGF (5 ng/mL). At specified time points, cells were subjected to morphological study, immunofluorescence staining, RT-PCR, LDL-uptake tests and 3-D culture for the examination of the structural and functional characteristics of endothelial cells. Classic cobblestone-like growth pattern appeared at 6 day of the induced differentiation. Immunofluorescence staining and RT-PCR analyses revealed that the induced cells exhibited endothelial cell-specific markers such as CD31, von Willebrand factor, Flk1, Flt1, VE-cadherin, Tie2, EphrinB2 and Vezf1 at 9 day. The induced C3H/10T1/2 cells exhibited functional characteristics of the mature endothelial phenotype, such as uptake of acetylated low-density lipoproteins (Ac-LDL) and formation of capillary-like structures in three-dimensional culture. At 9 day, Weibel-Palade bodies were observed under a transmission electron microscope. This study demonstrates, for the first time, endothelial differentiation of C3H/10T1/2 cells induced by angiogenic factors, VEGF and bFGF, and confirms the multipotential differentiation ability. This in vitro model is useful for investigating the molecular events in endothelial differentiation of mesenchymal stem cells.

[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.

[Experimental study on the myogenic differentiation of marrow mesenchymal stem cells in the local muscle tissues].

OBJECTIVE: To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. METHODS: The serious muscle-injured model was established by the way of radiation injury, incising, and freezing injury in 36 mice. Purified MSCs derived from bone marrow of male mouse and MSCs induced by 5-azacytidine (5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of female mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immunohistochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. RESULTS: The quantity of implanted MSCs decreased as time passed. MSCs' differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. CONCLUSION: MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiation occurs earlier in the induced MSCs group than that in purified MSCs group.

The effects of total-body irradiation on the survival and skin wound healing of rats with combined radiation-wound injury.

To investigate the effects of total body irradiation on the healing of skin wounds, rats were irradiated with a (60)Co gamma-ray source, in which single doses ranged from 1 to 8 Gy. After irradiation, two whole-thickness circular skin wounds, 22 mm in diameter and covering 2.5% of the total body surface area, were made immediately on the back of each animal. The average healing time for the simple wound was 18.3 +/- 2.1 days, whereas when the wound was combined with 1, 2, 3, 4, 5, and 6 Gy of radiation, the average wound healing time was delayed by 0.3, 0.8, 1.1, 3.5, 6.2, and 9.5 days, respectively. The average healing time was significantly decreased with irradiation doses exceeding 4 Gy, as compared with the healing time for the simple wound without irradiation (p < 0.05). The statistical results showed that the percentage of the unclosed wound with the increased doses in combined radiation injury was significantly proportional to the recovery time kinetics.

[Study on human amniotic membrane loaded with marrow mesenchymal stem cells and epidermis cells in promoting healing of wound combined with radiation injury].

OBJECTIVE: To investigate the results of human amniotic membrane (HAM) which are loaded with marrow mesenchymal stem cells (MSCs) and epidermis cells in treating full-thickness skin defect combined with radiation injury. METHODS: Eight minipigs were used in this study. Three round full-thickness wounds (phi3.67cm), which combined with radiation injury, were created on the dorsum of each side close to the vertebral column in each animal. Among 48 wounds, 24 left side wounds were treated with HAM loaded with MSCs and epidermis cells as experimental group (group A), 16 right side wounds with simple HAM (HAM group, group B) and 8 right side wounds with oil gauze as control (group C). The granulation tissue, reepithelization and wound area were observed after 1,2 and 3 weeks. Immunohistochemistry was performed using vWF as a marker for blood vessels. Image analysis was employed to test new area of wound at different interval time and healing rate of wound. RESULTS: The healing time of group A was 6 to 7 days faster than that of group C and 5 to 6 days fister than that of group B. After 15-17 days of graft, there were significant differences in new area of wound and healing rate between group A and groups B, C (P < 0. 01). New epidermis fully covered whole wound surface in group A, and their granulation tissue, which contained a lot of vWF, fibroblasts, capillaries and collagen, grew well. Many inflammatory cells still were seen in groups B and C, and their contents of vWF, fibroblasts, capillaries and collagen in granulation tissue were smaller than that in group A. CONCLUSION: The graft of HAM loaded with MSCs and epidermis cells played an effective role in promoting healing of wound combined radiation injury with high quality.

Reduced presence of tissue-repairing cells in wounds combined with whole-body irradiation injury is associated with both suppression of proliferation and increased apoptosis.

BACKGROUND: The goal of our study was to examine the effects of whole body irradiation injury on tissue-repairing cells, so as to elucidate the possible mechanisms of reduced repopulation of tissue-repairing cells. MATERIAL/METHODS: The quantity of tissue-repairing cells in wounds from simple incision injury (WBI) (group S) and combined with WBI injury (group C) was measured histologically. The proliferation and apoptosis of tissue-repairing cells in wounds were determined using immunohistochemical staining for proliferation cell nuclear antigen (PCAN) and by TUNEL assay. Fibroblasts were isolated from the wounds, and their proliferation and apoptosis were detected in vitro. Some key molecules involving in proliferation (cyclin E and cyclin dependent kinase 4) and apoptosis (bcl-2 and bax) were also determined, so as to interpret the mechanisms of the alteration of tissue repairing cells. RESULTS: The quantity of tissue-repairing cells in group C was significantly less than in group S. The PCNA contents in wounds of group C were significantly less than in group S, whereas the apoptosis of tissue-repairing cells in wounds of group C increased significantly as compared to group S. The expression of the molecules involved in the mediating of proliferation and apoptosis coincided with the proliferation and apoptosis of tissue repairing cells in wounds. CONCLUSIONS: The quantity of tissue repairing cells in wounds combined with WBI injury is significantly less than in simple incision injury. This is associated with both suppression of proliferation and increased apoptosis.

Dose-effect relationships in total body irradiation on the healing of cutaneous wounds.

OBJECTIVE: To study the effects of dosages of total body irradiation on the healing process of cutaneous wounds and to observe the changes of wound area at different periods after injury. METHODS: The entire body irradiation from a (60)Co gamma-ray source was performed on Wistar rats. The single dosage varied from 1 to 8 Gy. Within 1 h after irradiation, two whole thickness circular cutaneous wounds corresponding to 2.5% of total body surface area (Phi = 22 mm) were produced on the back of the animals (combined injury groups). Same wounds were produced on rats with no irradiation (single wound group). Wound healing was observed at different points after injury. RESULTS: After total body irradiation with the dose of 1, 2, 3, 4, 5, 6, 7 or 8 Gy, the wound healing was obviously retarded as the dosages increased. The wound area remained was larger in the large dosage groups than in the small dosage groups. Seven days after injury, there was 33.5% wound surface left unhealed in the single wound group, whereas in the combined injury groups, 35.4%, 38.1%, 41.6%, 48.8%, 53.9%, 63.7%, 69.2% and 73.9% of the wound surfaces remained unhealed, respectively. Statistical analysis showed marked correlations between the various times after total body irradiation and various dosages to the percentage of unhealed wound surface. Nine dose-effect relation formulae were deduced according to the statistical results. CONCLUSIONS: In soft tissue trauma combined with radiation injury, the delay of wound healing is related to the dose of radiation inflicted. It is also related to the time between injury and time of observation.

[The advances in the application of amniotic membrane stroma in promoting tissue repair].

Amniotic membrane is composed of amniotic epithelium, basement-membrane and stroma. Amniotic membrane is an easily obtained biomaterial and easily to be also processed, preserved and transported. However, its applicability will not be destroyed after it has been preserved for a long time(about one year). Thus it has been utilized widely in laboratory and clinical surgery. Generally, homologous amniotic membrane does not induce rejection after allotransplantation, and it is a bio-absorbable and degradable material. The purpose of this paper is to review the characteristics of amniotic membrane that makes it potentially useful in promoting tissue repair.

[The experimental study of bone marrow mesenchymal stem cells on the repair of skin wound combined with local radiation injury].

OBJECTIVE: To explore the effects of swine bone marrow derived mesenchymal stem cells (MSC) on the repair of skin wound combined local irradiation injury and theirs mechanism. METHODS: MSC were isolated from the adult swine bone marrow and autologous MSC were directly transplanted into the local site full thickness skin incision round wounds combined with 20 Gy irradiation. By means of laser confocal microscopy, quantitative collagen measurement, ELSIA, fibroblasts scrap test, RT-PCR technology and so on in order to observe the effects of MSC promote the repair of wound and theirs mechanism. RESULTS: Transplanted autologous MSC into local skin wound combined with 20 Gy irradiation could accelerate the speed of wound healing, the healing time moved up 3 - 4 days. The granule tissues were abundant in the wounds and the content of collagen was increased in MSC groups. On one hand, MSC attracted the inflammatory cells and repaired cells to immigrate into wounds by themselves or secretion of IL-6, IL-8 and G-CSF and thus initiated the repairing processes. On the other hand, the wound microenviroment induced the proliferation and changes of I collagen mRNA expression of MSC showed that MSC could secrete extracellular matrix or differentiate into fibroblast. CONCLUSION: Local transplanted MSC promoted the repair of skin wounds combined with irradiation injury. The promotion effects were achieved by the interaction between MSC and wound microenviroment.