Objective To investigate the application of fluorimetric reverse transcriptase-polymerase chain reaction(RT-PCR) to estimate postmortem interval(PMI)in rats.Methods A total of 44 rats were killed by cervical dislocation and placed in a chamber with temperature-controlling system at 20℃ for 0 to 8 d postmortem.Changes of the relative content of glyceraldehyde phosphate dehydrogenase(GAPDH) mRNA in the brain and liver were examined by fluorimetric RT-PCR.Results The amplification of GAPDH mRNA could be detected within 7 d in the brain and 2 d in the liver postmortem respectively,in a fashion of decreasing.Conclusion Fluorimetric RT-PCR can detect the time-dependent degradation of GAPDH mRNA in the brain and the liver,so it maybe provide a new index for estimating PMI.

Objective To study changes in expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the kidney and spleen of dead rats and its application in deducing the postmortem intervals (PMI), so as to look for a new method for PMI deduction. Method 28 rats were killed by cervical dislocation, placed in a chamber with temperature-controlling system at 20℃ for 0 to 48h postmortem, and autopsied at 0, 8, 16, 24, 32, 40 and 48h respectively (n=4 for each time point). The expression of GAPDH mRNA in kidney and spleen was assessed with fluorescence semi-quantitative RT-PCR. Results The amplified products of GAPDH mRNA could be detected in kidney within 40h and in spleen within 32h postmortem respectively, and they showed a decreasing tendency as its fluorescence intensity declined gradually and lost at last. Conclusion Detection of GAPDH mRNA expression in kidney and spleen of rats at different periods after death by fluorescence semi-quantitative RT-PCR seems to be of some value for estimation of PMI.

Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 micromol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.

Objective:To explore the expression of Nogo-A in membrane of mature oligodendrocyte, and obtain these living cells. Methods:Mature oligodendrocytes were stained by indirect immuno-fluorescent technique.Stained unpermeable oligodendrocytes were sorted by flow cytometry. Results: The percentage of mature oligodendrocytes with Nogo-A expression in membrane was 3.36%. Conclusion:Nogo-A could be expressed in membrane of mature oligodendrocytes,and the ratio of positive cells was about 3.36%.

This study investigated the effect of TNP-470 in combination with carmustine (BCNU) on the growth of subcutaneously implanted human glioblastoma xenografts in nude mice. Human glioblastoma U-251 cells (1×10(7)) were injected into 24 nude mice subcutaneously. The tumor-bearing mice were randomly divided into 4 groups on the seventh day following tumor implantation: TNP-470 group, in which TNP-470 was given 30 mg/kg subcutaneously every other day 7 times; BCNU group, in which 20 mg/kg BCNU were injected into peritoneal cavity per 4 days 3 times; TNP-470 plus BCNU group, in which TNP-470 and BCNU were coadministered in the same manner as in the TNP-470 group and the BCNU group; control group, in which the mice were given 0.2 mL of the mixture including 3% ethanol, 5% acacia and 0.9% saline subcutaneously every other day 7 times. The tumor size and weights were measured. The tumor microvessel density (MVD) was determined by immunostaining by using goat-anti-mouse polyclonal antibody CD105. The results showed that on the 21th day following treatment, the volume of xenografts in the TNP-470 plus BCNU group was (108.93±17.63)mm(3), markedly lower than that in the TNP-470 group [(576.10±114.29)mm(3)] and the BCNU group [(473.01±48.04)mm(3)] (both P<0.01). And the xenograft volume in these 3 treatment groups was even much lower than that in the control group [(1512.61±470.25) mm(3)] (all P<0.01). There was no significant difference in the volume of xenografts between the TNP-470 group and the BCNU group (P>0.05). The inhibition rate of the tumor growth in the TNP-470 plus BCNU group was (92.80±11.37)%, notably higher than that in the TNP-470 group [(61.91±6.29)%] and the BCNU group [(68.73±9.65)%] (both P<0.01) on the 21th day following treatment. There was no significant difference in the inhibition rate of tumor growth between the TNP-470 group and the BCNU group (P>0.05). The MVD of xenografts in the TNP-470 plus BCNU group was decreased significantly as compared with that in the TNP-470 group or the BCNU group (both P<0.05). The MVD of xenografts in the 3 treatment groups was markedly reduced as compared with that in the control group (all P<0.05). No significant changes in weights were observed before and after the treatment in each group (all P>0.05). It was concluded that the combination of TNP-470 and BCNU can significantly inhibit the growth of human glioblastoma xenografts in nude mice without evident side effects.

Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.

This study investigated the effect of TNP-470 in combination with carmustine (BCNU) on the growth of subcutaneously implanted human glioblastoma xenografts in nude mice. Human glioblastoma U-251 cells (1×10(7)) were injected into 24 nude mice subcutaneously. The tumor-bearing mice were randomly divided into 4 groups on the seventh day following tumor implantation: TNP-470 group, in which TNP-470 was given 30 mg/kg subcutaneously every other day 7 times; BCNU group, in which 20 mg/kg BCNU were injected into peritoneal cavity per 4 days 3 times; TNP-470 plus BCNU group, in which TNP-470 and BCNU were coadministered in the same manner as in the TNP-470 group and the BCNU group; control group, in which the mice were given 0.2 mL of the mixture including 3% ethanol, 5% acacia and 0.9% saline subcutaneously every other day 7 times. The tumor size and weights were measured. The tumor microvessel density (MVD) was determined by immunostaining by using goat-anti-mouse polyclonal antibody CD105. The results showed that on the 21th day following treatment, the volume of xenografts in the TNP-470 plus BCNU group was (108.93±17.63)mm(3), markedly lower than that in the TNP-470 group [(576.10±114.29)mm(3)] and the BCNU group [(473.01±48.04)mm(3)] (both P<0.01). And the xenograft volume in these 3 treatment groups was even much lower than that in the control group [(1512.61±470.25) mm(3)] (all P<0.01). There was no significant difference in the volume of xenografts between the TNP-470 group and the BCNU group (P>0.05). The inhibition rate of the tumor growth in the TNP-470 plus BCNU group was (92.80±11.37)%, notably higher than that in the TNP-470 group [(61.91±6.29)%] and the BCNU group [(68.73±9.65)%] (both P<0.01) on the 21th day following treatment. There was no significant difference in the inhibition rate of tumor growth between the TNP-470 group and the BCNU group (P>0.05). The MVD of xenografts in the TNP-470 plus BCNU group was decreased significantly as compared with that in the TNP-470 group or the BCNU group (both P<0.05). The MVD of xenografts in the 3 treatment groups was markedly reduced as compared with that in the control group (all P<0.05). No significant changes in weights were observed before and after the treatment in each group (all P>0.05). It was concluded that the combination of TNP-470 and BCNU can significantly inhibit the growth of human glioblastoma xenografts in nude mice without evident side effects.
Angiogenesis Inhibitors ; administration & dosage ; Animals ; Antibiotics, Antineoplastic ; administration & dosage ; Antineoplastic Agents, Alkylating ; administration & dosage ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Brain Neoplasms ; drug therapy ; Carmustine ; administration & dosage ; Cell Line, Tumor ; Cyclohexanes ; administration & dosage ; Female ; Glioblastoma ; drug therapy ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Sesquiterpenes ; administration & dosage ; Xenograft Model Antitumor Assays

Objective: To investigate the current status of the ten-year implementation of the World Health Organization (WHO) surgical safety checklist (SSC) in China. Methods: A questionnaire was designed based on the three phases described by the checklist — the period before induction of anaesthesia, the period before skin incision, and the period before patient leaves operating room, taking into account some hotspots and disputes.The questionnaire was sent to the members of the Chinese-based online New Youth Anesthesia Forum through the WeChat platform.Answers were completed by mobile phones or desktop computers.Each WeChat ID number allowed only one answer for each individual participant. Results: A total of 3 943 members red the questionnaire invitation, of which 2 121 members completed the questionnaire with an overall completion rate of 53.79%.For checks completed before induction of anesthesia, the percentage of members who routinely practiced checks before induction of anesthesia was 93.35%, the percentage of members who completed each and every element of the checklist was 60.16%, and the percentage of members who selected the element of the checklist the surgeon was not involved in the check was 14.05%.For checks practiced before skin incision, the percentage of members who routinely completed checks before skin incision was 78.22%, the percentage of members who completed each and every element of the checklist was 51.91%, and the percentage of members who selected the element of the checklist surgeons and anesthesiologists routinely stated their own professional key information was 18.24% and 18.81%, respectively.For checks practiced before the patient leaved the operating room, the percentage of members who routinely completed checks before removing the patient from the operating room was 64.26%, and the percentage of members who completed each and every element of the checklist was 44.18%.The percentage of members who was really serious about practicing the checklist was 56.20%.The percentage of members who believed that surgeons should participated in checks practiced before induction of anesthesia was 81.47%.If the member himself or a member of his family needed a surgery, the percentage of members who hoped to implement the checklist was 98.35%.The percentage of members who believed that practicing WHO SSC could reduce the complications of surgery and improve the anesthetic safety of patients was 94.34%. Conclusion The implementing rate of checks practiced before induction of anesthesia is high, while the implementing rates of checks completed before skin incision and before patient leaves operating room are sequentially reduced in China.Although there are some problems with the implementation of WHO SSC, most respondents believe that implementing SSC can improve the anesthetic safety of patients undergoing surgery.