1.Clinical application of the blood transfusion with equal ratio component in severe multiple injuries with acute traumatic coagulopathy
Shihua HU ; Wenxin JIANG ; Yanxia YANG ; Min SU ; Minghan DENG
Chongqing Medicine 2015;(1):68-70
Objective To investigate the clinical effect of the blood transfusion with equal ratio component in severe multiple in‐juries with acute traumatic coagulopathy(ATC) .Methods Thirty‐eight patients who had severe multiple injuries with ATC were divided randomly into control group and treatment group .Control group was treated with the different ratio packed red blood cells (PRBC)and fresh frozen plasma(FFP) ,while treatment group received the equal ratio PRBC and FFP .Hemoglobin(HB) ,pro‐thrombin time(PT) ,international normalized ratio(INR) ,fibrinogen(FIB)were measured on the 1st ,2nd ,3rd day after admission . The total amount of PRBC during these 3 days ,the days of hospitalization in ICU ,the corrected rate of shock ,the 28‐day mortality were compared between groups .Results Compared with the control group ,the levels of PT ,INR and FIB of treatment group on the 2nd ,3rd day after admission were better(P<0 .05) .The total amount of PRBC[(18 .5 ± 6 .3)U]during these 3 days ,the days of hospitalization in ICU [(5 .9 ± 4 .3)d] in treatment group were less than those in the control group [(25 .9 ± 7 .8)U ,(10 .5 ± 7 .6)d] (P<0 .05) ,while the corrected rate of shock(85 .0% )in treatment group was higher than that of the control group(44 .4% ) .The 28‐day mortality(10 .0% )in treatment group was lower than that of the control group(27 .8% )(P<0 .05) .Conclusion The blood transfusion with equal ratio component in severe multiple injuries with ATC could not only improve blood clotting index ,reduce the total amount of PRBC and the time in ICU ,but also increase the corrected rate of shock and decrease the 28‐day mortality .
2.The effect of cytoplasmic Ca2+ on cisplatin-induced autophagy in ovarian carcinoma SKOV3 cells and its mechanism
Linchuan ZENG ; Huimin DENG ; Jun CHEN ; Minghan DOU ; Ye XU
China Oncology 2016;26(4):313-319
Background and purpose:Ca2+plays a very important role in the maintenance of cell biological functions. The storage, release and uptake capacity of Ca2+ is controlled by endoplasmic reticulum (ER). Ca2+ homeo-stasis is essential for cellular energy metabolism and proper protein folding. This study aimed to investigate the effect of cytoplasmic Ca2+ on cisplatin induced ER stress-mediated autophagy in ovarian carcinoma SKOV3 and its underlying mechanism.Methods:The ovarian cancer SKOV3 was used as a study object. The experiment consisted of three parts:① To explore the possible relationship between cisplatin-induced ER stress and autophagy, SKOV3 cells were treated with cisplatin for 0, 6, 12 and 24 h, respectively;② To explore the possible relationship between ER stress induced Ca2+ effux and autophagy, SKOV3 cells were treated with cisplatin for 0, 9 and 12 h, respectively, and TG was used as a positive control;③ To explore the effects of blocking calcium effux on autophagy, SKOV3 cells were divided into control group, cisplatin group, TG group, BAPTA-AM group, cisplatin combined with BAPTA-AM group and TG com-bined with BAPTA-AM group. Western blot was used to detect the protein levels of GRP78 and LC3. Fluo-4 calcium lfuorescent probe was used to examine cytoplasmic Ca2+ levels. Confocal microscopy was used to detect LC3 level by immunolfurescence staining.Results:Compared to control group (0.679±0.011), GRP78 was signiifcantly accumulated at 6, 12 and 24 h after cisplatin treatment and reached the maximum value at 6 h (1.393±0.004,P=0.000). Similarly, compared to control group (0.038±0.000), LC3 puncta were clearly seen after cisplatin treatment and reached the maxi-mum value at 12 h (0.072±0.002,P=0.000). Using confocal microscopy, we found that cisplatin and TG increased LC3 punctate accumulation and cytoplasmic Ca2+ levels in a time-dependent manner. Immunolfuorescent method showed that treatment with cisplatin combined with BAPTA-AM or TG combined with BAPTA-AM increased LC3 punctate accumulation induced by cisplatin or TG. The results of Western blot showed that cisplatin combined with BAPTA-AM (0.071±0.001) or TG combined with BAPTA-AM (0.065±0.001) signiifcantly increased LC3Ⅱ/LC3Ⅰ ratio induced by cisplatin (0.039±0.000,P=0.000) or TG (0.035±0.001,P=0.000).Conclusion:Cisplatin induces intracellular ER stress and autophagy in SKOV3 cells, accompanied by increased cytoplasmic Ca2+ levels. Chelating cytoplasmic Ca2+enhanc-es cisplatin-induced autophagy.
3.Constructing the dual luciferase reporter vector containing human DRD1 promoter region
Chunhong WANG ; Zhe LI ; Minghan WANG ; Lili DENG ; Huan WANG
Chinese Journal of Tissue Engineering Research 2016;20(40):6060-6066
BACKGROUND:The polymorphisms of dopamine receptor in promoter region wil affect the expression of the receptor, thereby affecting the dopaminergic neurotransmitter, final y lead to related diseases.
OBJECTIVE:To construct the dual luciferase reporter vector containing human DRD1 promoter region and determine its activity, which could provide the basic tool for studying the transcriptional regulation of DRD1 gene.
METHODS:DRD1 promoter sequence was amplified by PCR using the human blood genomic DNA and cloned into pGM-T vector. After sequencing, the correctly constructed vectors were ligated to the firefly luciferase reporter plasmid pGL3-Basic. The cloned pGL3-Basic vectors were transfected into HEK293 using cationic liposome method. In the meanwhile, PGL3-Basic vector with no promoter was co-transfected with pGL3-TK plasmid as negative control group. The relative fluorescence intensity was measured by chemiluminescence.
RESULTS AND CONCLUSION:(1) Recombinant luciferase reporter gene vectors were confirmed by restriction analysis and sequencing. (2) Compared with the negative control group, the HEK293 cel s transfected by recombinant vectors presented transcriptional activity. (3) In conclusion, luciferase reporter gene vectors containing DRD1 promoter region are successful y constructed and can provide the basic tool for further study on the transcriptional regulation of DRD1.