Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment / 中国病理生理杂志

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model.Oil red O staining was used to determine whether the model was established successfully.miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h.The intracellular lipid droplets were observed by Oil red O staining.The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot.The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05).The intracellular cholesterol content was increased gradually (P<0.05) , and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group.Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05).No difference of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.

Identification of protein that interacts with adhesion protein of Mycoplasma genitalium from T7-phage display cDNA library / 中国免疫学杂志

Objective:To screen and identify the protein that interacts with the adhesion protein of Mycoplasma genitalium (MgPa)from T7-phage display cDNA library of human uroepithelial cells(SV-HUC-1).Methods:Recombinant adhesion protein of My-coplasma genitalium(rMgPa)was used as target molecule to biopan the T7 phage display cDNA library of SV-HUC-1 cell,the selected positive clones were analysed using DNA sequencing and BLAST analysis and identified by means of indirect ELISA,Dot immunoblot and Far-western blot.Results:After four rounds of biopanning,positive phages were obviously enriched.According to the results of DNA sequencing and BLAST analysis,the selected randomly 32 positive clones included 7 kinds different sequences,of which the number of RPL35 repeats was the most.The results of indirect ELISA,Dot immunoblot and Far-western blot showed that 7 representative phages could bind specifically with rMgPa.Conclusion:60S ribosomal protein L35(RPL35) may be the interacting protein of MgPa,which lays the experimental foundation for understanding the function of MgPa and the pathogenesis of Mycoplasma genitalium.

Facial pain induces the alteration of transient receptor potential vanilloid receptor 1 expression in rat trigeminal ganglion / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To investigate the involvement of transient receptor potential vanilloid receptor 1 (TRPV1) in the facial inflammatory pain in relation to thermal hyperalgesia and cold pain sensation.</p><p><b>METHODS</b>Facial inflammatory pain model was developed by subcutaneous injection of turpentine oil (TO) into rat facial area. Head withdrawal thermal latency (HWTL) and head withdrawal cold latency (HWCL) were measured once a day for 21 d after TO treatment using thermal and cold measurement apparatus. The immunohistochemical staining, cell-size frequency analysis and the survey of average optical density (OD) value were used to observe the changes of TRPV1 expression in the neurons of the trigeminal ganglion (TG), peripheral nerve fibers in the vibrissal pad, and central projection processes in the trigeminal sensory nuclei caudalis (Vc) on day 3, 5, 7, 14, and 21 after TO injection.</p><p><b>RESULTS</b>HWTL and HWCL decreased significantly from day 1 to day 14 after TO injection with the lowest value on day 5 and day 3, respectively, and both recovered on day 21. The number of TRPV1-labeled neurons increased remarkably from day 1 to day 14 with a peak on day 7, and returned back to the normal level on day 21. In control rats, only small and medium-sized TG neurons were immunoreactive (IR) to TRPV1, and the TRPV1-IR terminals were abundant in both the vibrissal pad and the Vc. Within 2 weeks of inflammation, the expression of TRPV1 in small and medium-sized TG neurons increased obviously. Also the TRPV1 stained terminals and fibers appeared more frequent and denser in both the vibrissal pad skin and throughout laminae I and the outer zone of laminae II (IIo) of Vc.</p><p><b>CONCLUSION</b>Facial inflammatory pain could induce hyperalgesia to noxious heat and cold stimuli, and result in increase of the numbers of TRPV1 positive TG neurons and the peripheral and central terminals of TG. These results suggest that the phenotypic changes of TRPV1 expression in small and medium-sized TG neurons and terminals might play an important role in the development and maintenance of TO-induced inflammatory thermal hyperalgesia and cold pain sensation.</p>

Changes of cyclin D1 expression following human brain contusion / 法医学杂志

OBJECTIVE@#To study the relationships of Cyclin D1 expression with the posttraumatic intervals (PTI) following the cerebra, brainstem or cerebella contusion in human.@*METHODS@#88 cases of brain contusions of the closed head injury were investigated with pathological and Cyclin D1 immunohistochemistry methods. The results were analyzed by image analysis technique (IAT).@*RESULTS@#The immunoreactivity of Cyclin D1 was almost disappeared in the core cells of the brain contusion. Cyclin D1-positive cells started to increase in the boundary of the brain contusion in the 1h group. Cyclin D1-positive cells were increased significantly in the 3 h-30 d groups and maintained at a high level in the boundary of the brain contusion of those groups. It is suggested that the Cyclin D1-positive cells were primarily origin from microglia and other glia. A few neurons expressed Cyclin D1.@*CONCLUSION@#Cyclin D1 can express in several kinds of brain cells following the contusion, especially in the glia cells. Cyclin D1-positive cells were increased obviously and rapidly after injury, so it could be used as a reference marker for early stage brain injury.

Relationship between expression of nestin in experimental brain contusion and injury time / 法医学杂志

OBJECTIVE@#To observe the alteration of nestin intervals in the experimental traumatic brain injury and investigate its relation to the injury intervals.@*METHODS@#The rat brain contusion was conducted by falling impact injury. After various survival interval (0.5, 6, 12 h and 1, 3, 7, 14, 28 d), immunohistochemical SP method was used for observing the expression of nestin in the cortex, hippocampal dentate gyrus and the corpus callosum on injury side.@*RESULTS@#Expression of nestin positive cells increased at 0.5 h and reached the maximum level in 7 d after brain contusion, then the expression decreased gradually. The intensity of nestin staining in the the cortex and the hippocampal dentate gyrus decreased to normal on 28 d. As to the corpus callosum of injury side it remained weak on 28 d.@*CONCLUSION@#The changes of nestin immunohistochemical staining can be used as an index for forensic estimation of early injury time.

The comparisons on total RNA from different source-original neurons applied in LMPC / 神经科学通报·英文版

Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods (1) Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12 h hypothalamus sections; (2) Different neurons of PVN and SON were collected by LMPC, CRH, TRH, AVP, OT mRNA level were measured by RT-PCR; (3) Labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR. Results (1) Unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC; relative short amplicons such as GAPDH, NSE, MCH and MC4R were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; (2) In mangocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two; (3) The expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons. Conclusion The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC and the CTB tracer which may differentiate neurons had no significant effect on physiology of the neurons applied in LMPC. The results showed that the LMPC technique is suitable for the qualitative and quantitative study on individual neurons at mRNA level.