BACKGROUND:Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation.
OBJECTIVE:To construct the targeting short hairpin RNA plasmid vector expressing TERT gene from astrocytes by using pLentilox3.7.U6.
METHODS:By using two sequences from TERT gene, we synthetized sense and antisense strand template sequences of RNA interference molecular in vitro, and then obtained the complementary strands through annealing procedure. We connected the strands with pLentilox3.7.U6 that was sequenced and transfected into the Escherichia coli. In the end, we tested its effect of reducing the TERT gene expressing by using cultured astrocytes from rat spinal cord in vitro through western blot and immunofluorescence technique.
RESULTS AND CONCLUSION:Western blot and immunofluorescence assay showed that, compared with the control group, the interference groups had a lower TERT expression in astrocytes. The targeting short hairpin RNA plasmid vector expressing TERT gene is useful to reduce the TERT gene expression. The targeting short hairpin RNA plasmid vector expressing TERT gene is valid for us to do the further test learning the mechanism of astrocytes in spinal cord injury.

Ten compounds were isolated from the 70% ethanol extract of linseed meal (Linum usitatissimum L) through a combination of various chromatographic techniques, including silica gel, macroporous adsorbent resin, Sephadex LH-20, and preparative HPLC. On the basis of spectroscopic data analysis, they were elucidated as 1-methylethyl-2-O-beta-D-glucopyranosyl-(1" --> 6')-beta-D-glucopyanoside (1), linustatin (2), neolinustatin (3), lotaustralin (4), linamarin (5), deoxyguanosine (6), deoxyadenosine (7), (+)-pinoresinol-4'-O-beta-D-glucopyranoside (8), 4-O-beta-D-glucopyranosylvanillyl alcohol (9) and tachioside (10), separately. Among them, compound 1 is a new compound, and compounds 6, 8 and 10 were isolated from the linseed meal for the first time.

BACKGROUND:Telomerase reverse transcriptase plays an important role in telomerase activation, and lentiviral vectors carrying human telomerase reverse transcriptase that can inhibit astrocyte expression are rarely reported to have effects on spinal cord injury. OBJECTIVE:To transfect rat astrocytes with telomerase reverse transcriptase gene lentiviral vectors and to observe the effect of telomerase reverse transcriptase gene lentiviral vectors on apoptosis of astrocytes. METHODS: Astrocytes from rats were subject to primary culture and subculture, and then transfected with siRNA lentiviral vector carrying telomerase reverse transcriptase gene (siRNA transfection group), simple lentiviral vector (lentiviral vector group) and nothing (blank group), respectively. Then, the transfection efficiency and apoptosis in different time periods after transfection were determined. RESULTS AND CONCLUSION:The transfection efficiency was up to 85%-90% after siRNA lentiviral vector and simple lentiviral vector transfection. The apoptosis rate of astrocytes in the siRNA transfection group was 50%-60% at 24-48 hours after transfection, but there was no significant change in the other two groups. These findings suggest that siRNA lentiviral vectors carrying telomerase reverse transcriptase gene can accelerate astrocytes apoptosis.

BACKGROUND:Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation, however there is rare report addressing the construction of the lentivirus targeted its genes to inhibit its expression in the spinal cord astrocytes.
OBJECTIVE:To construct recombinant lentivirus vector expressing smal interfering RNA against TERT gene and to evaluate its potential for inhibiting the TERT expression.
METHODS:After shRNA-TERT sequence was designed and synthesized, the sequence was amplified by PCR and then connected to plasmid pLentilox3.7U6-hTERT to construct recombinant plasmid. The recombinant plasmid was then transfected to DH5αcel s to screen positive colony, and the sequence was identified. The recombinant plasmid pLentilox3.7U6-TERT was transfected in 293T cel s, generating recombinant lentivirus Le-TERT. The titer of recombinant lentivirus was determined and Le-TERT was transfected into the rat spinal cord astrocytes. The expression of TERT in astrocytes was detected by RT-PCR, western blot and immunofluorescence assay.
RESULTS AND CONCLUSION:The gene sequencing analysis confirmed that, recombinant plasmid pLentilox3.7U6-TERT was successful y constructed. The real-time quantitative PCR, western blot analysis and immunofluorescence assay indicated that, after Le-TERT was transfected in the astrocytes for 4 days, the inhibition rate of TERT mRNA was (63.98±2.6)%, and Le-TERT was lowly expressed in the transfected astrocytes. Recombinant expression vector pLentilox3.7U6-TERT can produce the lentivirus at high titer and effectively inhibit TERT expression in the transfected astrocytes.

Objective: To construct the expression plasmid of a novel gene human NBEAL1 (neurobeachin like 1), and to study its relationship with the pathological grades of glioma. Methods: Total RNA of human glioma cell line U251 was extracted. NBEAL1 expression plasmid pGEX-KG/NBEAL1 was constructed and transferred into E. coli BL21. Recombinant NBEAL1 protein was induced by IPTG and further purified by GST affinity chromatographic column. The purity of recombinant NBEAL1 protein was examined by Western blotting analysis. A NBEAL1 protein specific monoclonal antibody was prepared and was used to study the relationship of NBEAL1 expression with pathological grades of glioma. Results: The NBEAL1 gene fragment was successfully cloned into pGEX-KG expression plasmid and verified by DNA sequencing. The recombinant NBEAL1 protein was expressed in inclusion bodies, with a yield of more than 30% of total bacterial proteins; the purity of purified NBEAL1 protein was above 95%. Western blotting analysis confirmed that the purified protein containing GST tag and NBEAL protein. NBEAL1 protein was lowly expressed in normal brain tissues and highly expressed in low grade glioma tissues; and the expression of NBEAL1 decreased with the increase of glioma malignancy. Conclusion: The NBEAL1 protein has been successfully cloned, expressed and purified. NBEAL1 protein expression in glioma tissues is negatively associated with the pathological grades of glioma.

Animals ; Blood Vessels ; pathology ; ultrastructure ; Cochlea ; blood supply ; Female ; Male ; Rats ; Rats, Inbred SHR ; Rats, Wistar

Animals ; Humans ; MicroRNAs ; physiology ; RNA, Small Interfering ; physiology ; RNA, Untranslated ; physiology

BACKGROUND: The study aimed to explore the effects of hypothermia state induced by 4 oC normal saline (NS) on liver biochemistry, enzymology and morphology after restoration of spontaneous circulation (ROSC) by cardiopulmonary resuscitation (CPR) in swine. METHODS: After 4 minutes of ventricular fibrillation (VF), standard CPR was carried out. Then the survivors were divided into two groups: low temperature group and normal temperature group. The low temperature (LT) group (n=5) received continuously 4 oC NS at the speed of 1.33 mL/kg per minute for 22 minutes, then at the speed lowering to 10 mL/kg per hour. The normal temperature (NT) group (n=5) received NS with normal room temperature at the same speed of the LT group. Hemodynamic status and oxygen metabolism were monitored and the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured in blood samples obtained at baseline and at 10 minutes, 2 hours and 4 hours after ROSC. At 24 hours after ROSC, the animals were killed and the liver was removed to determine the Na+-K+-ATPase and Ca2+-ATPase enzyme activities and histological changes under a light or electron microscope. RESULTS: Core temperature was decreased in the LT group (P<0.05), while HR, MAP and CPP were not significantly decreased (P>0.05) compared with the NT group (P>0.05). The oxygen extraction ratio was lower in the LT group than in the NT group (P<0.05). The serum levels of ALT, AST and LDH increased in both groups but not significantly in the LT group. The enzyme activity of liver ATP was much higher in the LT group (Na+-K+-ATP enzyme: 8.64±3.32 U vs. 3.28±0.71 U; Ca2+-ATP enzyme: 10.92±2.12 U vs. 2.75±0.78 U, P<0.05). The LT group showed less cellular edema, inflammation and few damaged mitochondria as compared with the NT group. CONCLUSION: These data suggested that infusing 4 oC NS continuously after ROSC could quickly lower the core body temperature, while maintaining a stable hemodynamic state and balancing oxygen metabolism, which protect the liver in terms of biochemistry, enzymology and histology after CPR.

Objective To analyze the miss rates of colorectal adenomas during colonoscopy as well as risk factors influencing the adenoma miss rates and to take corresponding measures. Methods A total of 432 patients who underwent index and follow-up colonoscopy in 18 months were randomized and investigated. The results of two colonoscopies were compared and the missed adenomas were defined as the adenomas de-tected only during the second colonoscopy. Miss rates were calculated according to patient-based methods. Chi-square test was used to analyze the relative factors influencing the adenoma miss rate of per-patient. Then the meaningful factors were chosen into the logistic regression model for multiple factors analysis. Results Of 432 patients,116(26. 9%)had missed adenomas on first colonoscopy. Single factor analysis found that the size of adenoma( χ2 = 89. 686,P = 0. 000),the shape of adenoma( χ2 = 68. 488,P = 0. 000),the location of adenoma(χ2 = 77. 055,P = 0. 000)and adenoma tissue types(χ2 = 417. 000,P = 0. 000)were the risk factors for miss rates of colorectal adenomas. Number of polyps(χ2 = 8. 450,P= 0. 038),the organi-zation type of polyp(χ2 = 10. 718,P= 0. 013)and proficiency of colonoscopists(χ2 = 56. 069,P= 0. 000), the quality of bowel preparation(χ2 = 39. 195,P = 0. 000),insertion time(χ2 = 13. 133,P = 0. 001)were also the risk factors for miss rates of colorectal adenomas. Logistic regression analysis showed that the bigger the adenoma size,the less missed adenomas(OR= 0. 341,95%CI:0. 173-0. 671). Also,the longer insertion time took,the lower the adenoma miss rate(OR = 0. 987,95% CI:0. 981-0. 994). Per-patient miss rates were lower for high-risk adenomas compared with low-risk adenomas(OR = 0. 324,95%CI:0. 154-0. 680). Adenomas happening in multiple parts of bowel easily leads to missing(OR= 3. 791,95%CI:1. 505-9. 546). Conclusion The missed diagnosis of adenomas is not only significantly associated with features of missed adenomas,but also with skills of colonoscopists,insertion time,and bowel preparation. The key is high-quality index colonoscopy to avoid adenomas missing.

BACKGROUND:A large number of studies have confirmed that anterior approach and posterior approach for multilevel cervical spondylotic myelopathy were effective, but there is stil no conclusion in which one is better.
OBJECTIVE:To systematical y assess the clinical effectiveness and safety of anterior approach versus posterior approach for multilevel cervical spondylotic myelopathy.
METHODS:The databases such as The Cochrane Library (Issue 3, 2013), PubMed (from 1966 to March 2013), OVID (from 1950 to March 2013), EMbase (from 1966 to March 2013), Chinese Biomedical Literature Database (from 1978 to March 2013), WanFang Database (from 1998 to March 2013), China National Knowledge Infrastructure (from 1999 to March 2013) were electronical y searched and five relevant journals were searched by hand to col ect the randomized control ed trials or non-randomized control ed trials about the clinical effectiveness and safety of anterior approach versus posterior approach for multilevel cervical spondylotic myelopathy. Two reviewers independently screened the literature according to the inclusive and exclusive criteria, extracted the data, and assessed the methodological quality of included studies. Then the meta-analysis was performed by using RevMan5.2 software.
RESULTS AND CONCLUSION:A total of 11 control ed trials involving 814 patients were included. Meta-analysis results showed that, compared with posterior approach, postoperative Japanese Orthopaedic Association scores were better (P<0.000 01), improvement rate of neurological function was higher (P=0.000 3), the incidence of C5 root palsy was lower (P=0.007), but operation time was longer (P<0.000 01), amount of intraoperative bleedin g was larger (P=0.000 7), incidence of adjacent segments degeneration was higher (P=0.01), incidence of postoperative complications was higher (P<0.000 01) and the rate of secondary surgical procedures was higher (P=0.003) after anterior approach. Additional y, there were no differences between the two groups in the cervical range of motion (P=0.56). For quantity limitation and low methodological quality of included studies, this conclusion stil needs to be further proved by performing more high-quality and large-scale randomized control ed trials.