Objective To construct lentiviral vector miR30-CD133 targeting CD133 gene and investigate its effects on SMMC7721 cells. Methods Thespecific sequence of DNA which containing both sense and antisense Oligo DNA of the targeting sequence were designed, synthesized and cloned into the pPRIME vector. pPRIME-miR30-CD133, psPAX2 and pMD2G were co-transfected on 293T cells to get the lentivirus the transfection efficiency was investigated by confocal laser scanning microscope. The expression of miR30-CD133 on CD133 were detected by qRT-PCR and Western blott. The proliferation and apoptosis effect of miR30-CD133 was respectively evaluated by CCK-8 assay and AnnexinV/PI. Results Functional PFU titers of PRIME-miR30-CD133 were 6.58 × 109 PFU/mL. The expression of CD133 mRNA in sh CD133 group (0.18 ± 0.04) was less than Blank group and shNC group (P<0.05). The expression of CD133 protein in sh CD133 group was less than Blank group (10%) and shNC group (8%) (P < 0.05). Cells proliferation activity were significantly inhibited in shCD133 group compared with control group. Apoptosis rate were significantly increased in shCD133 group (41.3%) compared with Blank group (25.3%)and shNC group (24.3%). Conclusion The lentivirus miR30 vector targeting CD133 gene was successfully constructed, which will be a basis to the further CD133 gene studies.

Objective To study the effects of lentiviral vector of microRNA targeting IGF1R gene on growth of liver cancer cells.Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pPRIME vector,named pPRIME-IGFIR-miR30-shRNA.The viruses were propagated on 293T cells.Viruses were purified by CsCI gradient according to standard techniques,and functional PFU titers were determined by plaque assay on 293 cells.The effect of pPRIME-IGFIR-miR30-shRNA on IGFIR expression of Hep3B、SMMC7721 cells was detected by RT-PCR and Western blot.The antitumor potential of PRIME-IGFIR-miR30-shRNA to Hep3B、SMMC7721 cells was evaluated by CCK-8 assay and Tunel.Results PRIME-IGFIR-miR30-shRNA was constructed successfully.Functional PFU titers of pPRIME-IGFIR-miR30-shRNA were 4.58?109PFU/ml.PRIME-IGFIR-miR30-shRNA inhibited IGFIR expression and the proliferation of Hep3B、SMMC7721 cells.Conclusions PRIME-IGFIR-miR30-shRNA expressing IGFIR-siRNA can inhibit IGFIR expression and may be used for further investigation of gene therapy of liver cancer.

Air Pollution ; analysis ; Barium ; analysis ; chemistry ; Solubility ; Spectrophotometry, Atomic ; methods ; Workplace

Adult ; Breast Neoplasms ; diagnostic imaging ; radiotherapy ; surgery ; Cone-Beam Computed Tomography ; Female ; Humans ; Mastectomy, Segmental ; Postoperative Period ; Radiotherapy Planning, Computer-Assisted ; methods ; Respiration

Objective To observe the influence of hIL-10-MSCs on rejection of rat discordant liver xenotransplantation. Methods The model of cavia to rat discordant liver xenotransplantation was established. The rats were randomized into the control group, MSCs group, and hIL-10-MSCs group.The survival time and liver function of each recipient were observed and expression levels of E-Selectin, LFA-1, VCAM-1, and NF-κB determined by RT-PCR and ELISA 24 h after transplantation.Results The survival time was prolonged, liver function improved, and expressions levels of E-Selectin, LFA-1, VCAM-1 and NF-κB were significantly decreased in the hIL-10-MSCs group (P＜0.05).Conclusion hIL-10-MSCs are able to protect transplanted liver and decrease the expression of E-Selectin, LFA-1, VCAM-1, and NF-κB in the liver.

Objective To investigate the effects of alcohol on hippocampal apoptosis-inducing factor (AIF) and caspase-3 expression in cercbral ischemia/reperfusion in rats.Methods Sixty-eight healthy adult male Wistar rats were randomly divided into a sham operation group (n =4) and a saline control group as well as low-dose (1.0 g/kg),medium-dose (1.5 g/kg) and high-dose (2.0 g/kg) ethanol groups.The saline control group and each dose alcohol group were redivided into ischemia/reperfusion 0,1,2 and 3 h subgroups according to the intervention time points (n =4 in each group).A model of middle cerebral artery ischemia/reperfusion in rats was induced by the suture method.The corresponding doses of ethanol or an equal volume of saline were injected intraperitoneally at ischemia for 2 h and reperfusion for 0,1,2 and 3 h in the alcohol groups and the saline control group.At ischemia for 2 hand reperfusion for 24 h,the neurological deficit in rats was evaluated by using behavioral score.Immunohistochemistry assay was used to detect the expressions of AIF and caspase3 in the hippocampus of ischemic sides at ischemia for 2 hand reperfusion for 24 h.Results The behavior evaluation showed that the neurological deficit score was 0 in the sham operation group.The neurological deficit scores in the different dose ethanol groups were significantly lower than those in the saline control group at the same intervention time points (all P=0.000),and there were significant differences between the same intervention time points in the different dose ethanol groups (all P =0.000).The high-dose ethanol group was the lowest.There were no significant differences between the different intervention time points in the same dose ethanol groups (all P＞0.05).Immunohistochemistry revealed that the numbers of positive cells of AIF and caspase-3 in the sham operation group were 17.21 ±2.86 and 20.60 ±4.39,respectively,and they were significantly less than those in the saline control group and the each dose ethanol group at ischemia/reperfusion 0 h (all P =0.000); the number of positive cells of hippocampal AIF and caspase-3 in the different dose ethanol groups were all significantly less than those in the saline control group at the same intervention time points (all P =0.000).There were significant differences between the same intervention time points in the different dose ethanol groups (all P =0.000),and the high-does ethanol group was the lowest.There were no significant differences between the different intervention time points in the same dose ethanol groups (all P =0.000).Conclusions Alcohol has protective effect on the cerebral tissue in ischemia/reperfusion injury in rats.Its mechanism may be associated with the inhibition of AIF and caspase-3 expression.

Objective To observe the effects of hIL-10-MSCs on heterologous T cell prolifera-tion. Methods hlL-10 was cloned by RT-PCR and then hIL-10/pLOX-cwGFPs was construct. The lentivirus was transfected into cavy MSCs and cell culture supernatant was tested for IL-10 protein by ELISA assay. The effects of hIL-10-MSCs on heterologous T cell proliferation were determined by CCK-8. The effect of peripheral blood mononuclearcell secretion IFN-γ was tested by EIASA. Results hIL-10/pLOX-cwGFP were constructed successfully and hIL-10-MSCs cell culture supernatant of IL-10 protein were increased obviously, hIL-10-MSCs could inhibit heterologous T cell proliferation sig-nificantly(P<0.05) but could not induce T cell proliferation (P<0.05). Adding IL-2 could reverse this inhibition(P<0.05), hIL-10-MSCs cell culture supernatant could inhibit peripheral blood mono-nuclearcell secretion IFN-γ. Conclusion hIL-10-MSCs may play an important role in significant im-munosuppression of heterologous T cell proliferation and suppression of secretion IFN-γ by peripheral blood mononuclear cells.

Breast Neoplasms ; diagnostic imaging ; radiotherapy ; Female ; Humans ; Imaging, Three-Dimensional ; Mastectomy, Segmental ; Postoperative Period ; Radiotherapy Planning, Computer-Assisted ; methods ; Respiration ; Surgical Instruments ; Tomography, X-Ray Computed ; methods ; Tumor Burden

Humans ; Imaging, Three-Dimensional ; Neoplasms ; radiotherapy ; surgery ; Radiosurgery ; instrumentation ; methods ; Tomography, X-Ray Computed

Objective To observe the influence of hIL-10-MSCs on apoptosis of rats subject to discordant liver xenotransplantation. Methods The model of guinea pig to rat discordant liver xenotransplantation was set up. The orthotopic liver transplantation model was established by using modified two-cuff technique. Following groups were designed: control group, MSCs group and hIL-10MSCs group. Before and after operation, the recipients in control group were injected with normal sodium (2 ml) and decaesadril (0.5 ml) ;Those in MSCs group were injected with MSCs (4.0× 106/ml)and decaesadril (0.5 ml); Those in hIL-10-MSCs group were injected with hIL-10-MSCs (4.0 × 106/ ml)and decaesadril (0.5 ml). Twelve h after operation, the livers of recipient were observed by HE staining and electron microscope, and apoptosis of the livers was detected by using TUNEL. The expression levels of hIL-10, caspase-3, Fas and FasL were assayed by Western blot or immunohistochemistry. Results As compared with control group and MSCs group, the pathological changes was alleviated, the expression of IL-10 was significantly increased, the expression of FasL was significantly reduced in hIL-10-MSCs group. The positive area size of Fas and Caspase-3 in hIL-10MSCs group was 11.5 % and 25.1 %, respectively, significantly smaller than those in control group (35.3 % and 70.8 % respectively, P＜0.05. Apoptosis index in hIL-10-MSCs group was 32.5 %,which was significantly lower than in control group (74.1%) and MSCs group (50. 3 % ). ConclusionhIL-10-MSCs can notably decrease apoptosis of the transplanted liver probably by inhibiting theexpression of Fas/FasL.