An intervention study of differential expressions of miRNA-21 in rats with traumatic brain injury / 中华神经医学杂志

Objective To evaluate the dynamic changes ofmiRNA-21 expression in injured brain tissues of rats with traumatic brain injury (TBI),and explore the effect of ectogenic miRNA-21 on neuronal apoptosis of the rats and their neurological functions.Methods Eighty-four rats were randomly divided into sham-operated group,TBI model group,blank-intervention group and miRNA-21-intervention group (n=21).Rats in the sham-operated group were only performed scalp incision and window bone removal without beating,and those in the other three groups were performed beating to induce TBI models; liposomes with/without miRNA-21 were injected into the latter two groups.Several time points after the brain injury/intervention:12,24,48 and 72 h,and 1 and 2 weeks,respectively,were chosen to measure the neurological functions using Modified Neurological Severity Scale (mNSS) and footfault test.Then,the animals were sacrificed to observe the miRNA-21 expression levels by using quantitative real-time-PCR and to examine the neuronal apoptosis in rat cerebral corticesby TUNEL.Results The miRNA-21 expression began to obviously increase in injured brain tissues at 2 h after TBI,peaked at 48 h after TBI in the cerebral cortices; the miRNA-21 expression level was still higher than that in the sham-operated group 7 d after TBI (P＜0.05); the miRNA-21 expression level was higher than that in the TBI model group and blank-intervention group at 24,48 and 72 h after TBI (P＜0.05).Began with 24 h of TBI,mNSS showed that the scores of miRNA-21-intervenion group were significantly lower than those in the TBI model group and blank-intervention (P＜0.05).TUNEL indicated that the count of apoptotic cells in the traumatic area of miRNA-21-intervenion group was significantly smaller than that in the TBI model group (P＜0.05) Conclusion MiRNA-21 may be involved in the process of recovery after traumatic brain injury to inhibit the apoptosis in the traumatic area.

Triterpenoids from Inonotus obliquus protect mice against oxidative damage induced by CCl4 / 药学学报

To investigate the effects of lanosterol (1), inotodiol (2) and trametenolic acid (3) from Inonotus obliquus against oxidative damage induced by CCl4 in mice, 1, 2 and 3 (20, 10 and 5 mg x kg(-1)) were respectively administered to mice, once a day for 3 days. Then the mice were induced to oxidative damage by CCl4 on the third day 30 min after the administration. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) and the content of malondialdehyde (MDA) and reductive glutathione (GSH) in serum and liver homogenate were determined. And the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and interleukin-6 (IL-6) concentration in serum were detected. The results showed that treatment with compound 1, 2 and 3 could significantly increase the activities of SOD, CAT and GSH-PX in serum and liver homogenate. Furthermore, the content of GSH in serum and liver homogenate increased and MDA content decreased markedly. In addition, compound 1, 2 and 3 could significantly inhibit the activities of ALT and AST in serum, and decrease the IL-6 concentration in serum remarkably. So, compound 1, 2 and 3 can protect mice against oxidative stress injury induced by CCl4. Furthermore, compound 1, 2 and 3 can protect cells from damage through inhibition on ALT, AST and the expression of IL-6.

Construction of recombinant adeno-associated virus vector rAAV2-PD-L1 and its biological efficiency in transfecting mouse vascular endothelial cells / 第二军医大学学报

Objective: To construct a recombinant adeno-associated virus vector rAAV2-PD-L1 and to investigate the biological efficiency of rAAV2-PD-L1-transfected vascular endothelial cells in co-stimulating secretion of cytokines by T cells. Methods: Mouse PD-L1 cDNA was amplified by RT-PCR from total RNA of mouse liver tissues and was cloned into shuttle vector pSNAV1; the products were then transferred into BHK21 cells by lipofectamine and rAAV2-PD-L1 was screened out. Mouse vascular endothelial cell line 2F-2B was infected with rAAV2-PD-L1 and were co-cultured with activated mouse T cells, and the IFNγ content was identified by ELISA in the supernatant. Results: The sequences of PD-L1 cDNA and pSNAV-PD-L1 were confirmed to be correct. The recombinant rAAV2-PD-L1 was verified by PCR and SDS-PAGE analysis. The virus physical titer was 4×1012 virus genome/ml and the protein concentration was 0.355 mg/ml. There was a high expression of PD-L1 in mouse vascular endothelial cells infected with rAAV2-PD-L1. The content of IFNγ in the culture supernatant was significantly decreased 48 hours after co-culture. Conclusion. The recombinant rAAV2-PD-L1 can infect vascular endothelial cells and inhibit secretion of IFNγ by activated T cells through co-stimulation.

The specific cytotoxic effect of tumor infiltrating lymphocytes transfected with chimeric T cell receptor on cells which express KDR / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the specific cytotoxity of tumor infiltrating lymphocytes (TIL) transfected with chimeric T cell receptor (CTCR) on cells which express KDR.</p><p><b>METHODS</b>A recombinant retroviral plasmid (pMSCVneo-Vhgamma) was constructed by cloning VEGF121-hinger-FcRgamma (Vhgamma) into retroviral vector pMSCVneo. After packaging by PT67, the virus with high titer was used to infect TIL isolated from liver cancer tissues, and then MSCVneo-Vhgamma-TIL was generated. TIL infected with MSCVneo was used as a control. The cytotoxicty of the transgenic TIL on NIH3T3 and HepG2 expressing no KDR and on ECV304 and A375 expressing KDR was detected with MTT colorimetric assay.</p><p><b>RESULTS</b>The sequences of VEGF121 and hinger-FcRgamma were different from those reported, but the deduced amino sequences were identical to the reported ones. The cytotoxity of TIL infected with MSCVneo on target cell was similar to that of the control TIL; both only had mild cytotoxity on cancer cell line. No significant cytotoxity was found in TIL infected with MSCVneo-cTCR on NIH3T3, but its cytotoxity on ECV304 was significant. The cytotoxity on HepG2 was similar to that of MSCVneo-TIL and uninfected TIL, but cytotoxity on A375 was significantly higher.</p><p><b>CONCLUSION</b>Chimeric T cell receptor permanently grafts TIL cell with predefined new specificity. TIL expressing Vhgamma can selectively recognize and kill vascular endothelial cell and tumor cells which express VEGF receptor KDR.</p>

Correlation between serum alpha-fetoprotein variant levels in patients with hepatocellular carcinoma and the presence of alpha-fetoprotein mRNA in the circulating blood / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between serum alpha-fetoprotein (AFP) variant levels in patients with hepatocellular carcinoma (HCC) and cancer cells disseminating through blood.</p><p><b>METHODS</b>Serum AFP variant levels were measured by crossed immunoaffino-electrophoresis in the presence of lectin before initial surgical treatment in HCC patients. Circulating tumor cells were simultaneously detected in pre-operative blood samples using reverse transcription-polymerase chain reaction (RT-PCR) for AFP mRNA.</p><p><b>RESULTS</b>Forty-six HCC patients with serum AFP positive were studied. Serum AFP variant level > or 20% was showed in 37 patients, among whom there were 22 (59.5%) showing AFP mRNA positive. In contrast, the positive AFP mRNA expression was only observed in 2 out of 9 patients (22.2%) with AFP variant level<20% (x(2)=4.02, P<0.05).</p><p><b>CONCLUSION</b>In hepatocellular carcinoma patients, increased AFP variant levels are associated with a haematogenous spread of tumor cells.</p>