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OBJECTIVE@#To investigate the effects of mTOR inhibitors everolimus (EVE) and gemcitabine (GEM) on the proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma (DLBCL) cell line U2932, and further explore the molecular mechanisms, so as to provide new ideas and experimental basis for the clinical treatment of DLBCL.@*METHODS@#The effect of EVE and GEM on the proliferation of U2932 cells was detected by CCK-8 assay, the IC50 of the two drugs was calculated, and the combination index (CI=) of the two drugs was calculated by CompuSyn software. The effect of EVE and GEM on apoptosis of U2932 cells was detected by flow cytometry with AnnexinV-FITC/PI staining. Flow cytometry with propidium iodide (PI) staining was used to detect the effect of EVE and GEM on the cell cycle of U2932 cells. Western blot assay was used to detect the effects of EVE and GEM on the channel proteins p-mTOR and p-4EBP1, the anti-apoptotic proteins MCL-1 and Survivin, and the cell cycle protein Cyclin D1.@*RESULTS@#Both EVE and GEM could significantly inhitbit the proliferation of U2932 cells in a time- and dose-dependent manner (r=0.465, 0.848; 0.555, 0.796). According to the calculation of CompuSyn software, EVE combined with GEM inhibited the proliferation of U2932 cells at 24, 48 and 72 h with CI=<1, which had a synergistic effect. After treated U2932 cells with 10 nmol/L EVE, 250 nmol/L GEM alone and in combination for 48 h, both EVE and GEM induced apoptosis, and the difference was statistically significant compared with the control group (P<0.05). The apoptosis rate was significantly enhanced after EVE in combination with GEM compared with single-agent (P<0.05). Both EVE and GEM alone and in combination significantly increased the proportion of cells in G1 phase compared with the control group (P<0.05). The proportion of cells in G1 phase was significantly increased when the two drugs were combined (P<0.05). The expression of p-mTOR and effector protein p-4EBP1 was significantly downregulated in the EVE combined with GEM group, the expression of anti-apoptotic proteins MCL-1, Survivin and cell cycle protein cyclin D1 was downregulated too (P<0.05).@*CONCLUSION@#EVE combined with GEM can synergistically inhibit the proliferation of U2932 cells, and the mechanism may be that they can synergistically induce apoptosis by downregulating the expression of MCL-1 and Survivin proteins and block the cell cycle progression by downregulating the expression of Cyclin D1.
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Humans , Gemcitabine , Everolimus/pharmacology , Survivin/pharmacology , Cyclin D1/pharmacology , Myeloid Cell Leukemia Sequence 1 Protein , Cell Line, Tumor , Cell Proliferation , TOR Serine-Threonine Kinases , Apoptosis , Apoptosis Regulatory Proteins , Cell Cycle Proteins , Lymphoma, Large B-Cell, DiffuseABSTRACT
This paper aims to investigate the protective effect and mechanism of Astragalus membranaceus and Angelica sinensis before and after compatibility against triptolide(TP)-induced hepatotoxicity. The experiment was divided into a blank group, model group, Astragalus membranaceus group, Angelica sinensis group, and compatibility groups with Astragalus membranaceus/Angelica sinensis ratio of 1∶1, 2∶1, and 5∶1. TP-induced hepatotoxicity model was established, and corresponding drug intervention was carried out. The levels of alanine transaminase(ALT), aspartate transaminase(AST), and alkaline phosphatase(ALP) in serum were detected. Pathological injuries of livers were detected by hematoxylin-eosin(HE) staining. The levels of malondialdehyde(MDA), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), and reduced glutathione(GSH) in the liver were measured. Wes-tern blot method was used to detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(Keap1), peroxisome proliferator-activated receptor gamma, coactivator-1 alpha(PGC-1α), heme oxygenase-1(HO-1), and NAD(P)H quinone dehydrogenase 1(NQO1) in livers. Immunofluorescence was used to detect the expression of Nrf2 and PGC-1α in livers. The results indicated that Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 could significantly reduce the levels of serum AST, ALT, and ALP, improve the pathological damage of liver tissue, increase the levels of GSH and GSH-Px, and reduce the content of MDA in liver tissue. Astragalus membranaceus/Angelica sinensis ratio of 1∶1 and 2∶1 could significantly improve the level of SOD. Astragalus membranaceus and Angelica sinensis before and after compatibility significantly increased the protein expression of HO-1 and NQO1, improved the protein expression of Nrf2 and PGC-1α, and decreased the protein expression of Keap1 in liver tissue. The above results confirmed that the compatibility of Astragalus membranaceus and Angelica sinensis had antioxidant effects by re-gulating Keap1/Nrf2/PGC-1α, and the Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 had stronger antioxidant effect and significantly reduced TP-induced hepatoto-xicity.
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Humans , Astragalus propinquus , Angelica sinensis , NF-E2-Related Factor 2/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , Antioxidants/pharmacology , Chemical and Drug Induced Liver Injury/prevention & control , Superoxide Dismutase/metabolism , Oxidative Stress , Diterpenes , Epoxy Compounds , PhenanthrenesABSTRACT
Objective: To discuss the effect of Dazhu Hongjingtian capsule to unstable carotid plaque (Qi deficiency and blood stasis syndrome), and its mechanisms in resisting oxidation, inflammation and thrombosis. Method: One hundred and twenty-three patients were randomly divided into control group (61 cases) and observation group (62 cases) by random number table. Patients in control group got aspirin enteric-coated tablets, 0.1 g/time, 1 time/day, and rosuvastatin calcium capsules, 10 mg/time, 1 time/day. In addition to the therapy of control group, patients in observation group were also given Dazhu Hongjingtian capsule, 4 grains/days, 3 times/days. And the outpatient follow-up was made once every 2 weeks for continued 16 weeks. Intima-media wall thickness (IMT), and number and size of plaque were detected by color Doppler ultrasound. And levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), superoxide dismutase (SOD), malondialdehyde (MDA), hypersensitive C reactive protein (hs-CRP), homocysteine (Hcy), matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), platelet alpha granular membrane protein (GMP-140), P-selectin on surface of platelets (CD62 p), lysosome glycoprotein (CD63), and vascular pseudotumor factor (VWF) were detected. Qi deficiency and blood stasis syndrome were graded. Result: After treatment, IMT in observation group was thinner than that in control group, number of plaque was less than that in control group, and size of plaque was smaller than those in control group (PPPPConclusion: In addition to the treatment for resisting platelet and regulating blood lipid, Dazhu Hongjingtian capsule can lessen and reduce arterial speckle regulate lipid metabolism, ameliorate symptoms of traditional Chinese medical (TCM) syndrome, with effects in resisting oxidation, inflammation and thrombotic formation.
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@#Objective To investigate the effects of transcranial direct current stimulation (tDCS) on motor function of upper limbs of stroke patients. Methods 80 stroke patients were randomly divided into experimental group and control group. Both groups accepted routine rehabilitation, while the experimental group accepted anodal stimulation, and the control group received sham stimulation. They were assessed with Brunnstrom stages of arms and hands, Fugl-Meyer Assessment (FMA) of upper extremities, Action Research Arm Test (ARAT), Motor Assessment Scale (MAS) and modified Barthel Index (MBI) before and 1 month after treatment. Results All the scores improved in both groups after treatment (P<0.05), and improved more in the Brunnstrom stages of arms and hands, FMA, ARAT in the experimental group than in the control group (P<0.05). Conclusion tDCS may promote the recovery of arms and hands function of stroke patients.
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Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem ( ES) cells.Meth-ods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6( Cg)-Tyrc-2J and BALB/c mouse blastocysts, respectively.The efficiency was statistically evaluated according to three aspects:blastocyst collection, chimera production and germline transmission.Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyrc-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts.Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P =0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice ( P<0.01).Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse.Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.
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Objective To discuss the effect of in vitro fertilization ( IVF) and mouse sperm cryopreservation , to establish a simple and economic frozen system for the genetically engineering mice preservation .Methods Sperm from genetically engineering mice were cryopreserved , IVF was performed using post-thawed sperm, then embryo transfer, to compare the effects of cryopreservation medium、age of male mice and sperm preincubation medium .Results Using CPA as sperm cryopreservation medium , when PM was used thawed-sperm preincubation in IVF , the fertility rates were from 82.49%to 91.43%, when HTF was used thawed-sperm preincubation in IVF , the fertility rates were from14.46%to 27.38%, there was a signification difference between PM and HTF sperm preincubation medium;10 to 35 weeks male genetically engineering mice sperm were succeed cryopreservation , and positive mice were procreated after 2-cell embryos were transferred;R18S3、CPM and CPA was used to freeze sperm , the fertility rates were 75.85%、88.89%to 94.27%, positive mice were procreated after 2-cell embryos were transferred;2-cell embryos after IVF were freezed , then thawed and positive mice were procreated after 2-cell embryos were transferred .Conclusion Using CPA as sperm cryopreservation medium , when PM was used thawed-sperm preincubation in IVF , genetically engineering mice sperm were succeed cryopreservation .
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Objective To obtain the basic growth parameters of a self-established F1 hybrid CB6F1 C57-ras transgenic mouse model, and to provide basic information for commercialization of this mouse model. Methods F1 hybrid mice (CB6F1) were produced by crossing C57-ras heterozygous transgenic (c-Ha-ras+/-) male mice and wild-type BALB/cJ female mice.The average litter size, weaning rate, sex ratio, growth performance and C57-ras transgenic positive rate were recorded and analyzed.Results The average litter size was eight, weaning rate was 90%, and sex ratio was approximately in accordance with prediction.The average body weight of newborn mice was 1.73 ±0.05 g.The average body weight of 10-week old c-Ha-ras transgenic female and male mice in CB6F1 background was 24.38 ±1.74 g and 29.42 ±1.72g, respectively, which had a significant difference (P<0.01).The c-Ha-ras transgenic positive rate was 46.9%. which was in accordance with genetic rules.Conclusions The F1 hybrid mice (CB6F1) produced by crossing C57-ras heterozygous transgenic ( c-Ha-ras +/-) male mice and wild-type BALB/cJ female mice show normal growth performance and development characteristics, and it can be used for large-scale commercial supply.
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Vitrification provides a rapid cooling to induce glass-like solidification inside cells and protect cell membrane and cytoskeleton system free of injury by ice crystals.The main factors that can influence the effect of vitrification are the size of ovarian tissue, the kind of cryoprotectants, the method of permeation, carrier system and so on. Ovarian tissue structure is very complex, and there is no uniform standardized protocol of vitrification yet.This paper presents also the current problems of ovarian tissue vitrification.
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Objective To establish a simple , fast and economic total DNA extraction method to serve the rapid identification of model mouse genotype in large number of mice .Methods Three methods, i.e.phenol extraction, isopropyl alcohol precipitation and mouse ear boiling methods were used to extract the total DNA from ten C 57-rasmodel mice.The purity and yield of DNA obtained by the three methods were compared , and polymerase chain reaction ( PCR) assay was used to compare the efficacy of the three extraction methods .Results Among the three methods , phenol extraction was the best and isopropyl alcohol precipitation was the poorest in DNA yield .In terms of DNA purity , the phenol extraction was the best and the mouse ear boiling method was the poorest .All the three methods could be used to extract the total DNA from mice serving as template of PCR reaction for the mouse genotype identification .The time consumption of three methods are 12.5 hr ,13 hr and 0.18 hr.Mouse ear boiling method was significantly lower than that of the other two methods ( P <0.01 ) ,.The obtained total DNA can be stored at conventional -20℃for 7 days and 30 days later still can be used as a template for PCR reaction .Conclusions Among the three methods studied , the mouse ear boiling method is simple and with the lowest cost , so it is feasible for total DNA extraction in scaled genotyping experiments .
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<p><b>OBJECTIVE</b>To observe the effects of scorpion and centipede on interleukin (IL)-2, IL-4, IL-10 in the small intestinal mucosa and joint injury of rats with collagen induced arthritis (CIA).</p><p><b>METHODS</b>Sixty Wistar rats were randomly divided into 6 groups: the normal control group, the model group, the low dose scorpion and centipede group, the middle dose scorpion and centipede group, the high dose scorpion and centipede group, and the type II collagen treatment group. The joints' volume was measured 40 days after type II collagen (CII) induced rheumatoid arthritis model was established. The joint injury was observed by naked eyes. The expression levels of IL-2, IL-4, and IL-10 in the small intestine tissue homogenate were detected by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The joint injury score and volume of two hind limbs were obviously higher in the model group than in the normal control group since the 23rd day (P < 0.01). Rats were accompanied with red, swollen, and deformed foot toes and ankle joints. Walking was even affected. Meanwhile, the joint injury score and volume of two hind limbs were obviously lowered by medicated with 0.4, 0.2, 0.1 g/kg scorpion and centipede, as well as CII on the 32nd day after medication (P <0.05, P < 0.01). The expression levels of IL-2, IL-4, and IL-10 in the small intestine tissue homogenate were obviously lower in the model group than in the normal control group (P < 0.05, P < 0.01). Compared with the model group, only the expression levels of IL-2 and IL-4 in the small intestine tissue homogenate of the high dose scorpion and centipede group and the type II collagen treatment group significantly increased. The expression level of IL-10 significantly increased in the high and middle dose scorpion and centipede groups, as well as the type II collagen treatment group, showing statistical difference (P < 0.05, P < 0.01).</p><p><b>CONCLUSIONS</b>Scorpion and centipede could effectively release the joint injury of rats with CIA. Its mechanism might be correlated with increased expression levels of IL-2, IL-4, and IL-10 in the small intestine mucosa.</p>
Subject(s)
Animals , Female , Rats , Arthritis, Experimental , Drug Therapy , Metabolism , Pathology , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Interleukin-10 , Metabolism , Interleukin-2 , Metabolism , Interleukin-4 , Metabolism , Intestinal Mucosa , Metabolism , Intestine, Small , Metabolism , Joints , Metabolism , Pathology , Rats, Wistar , ScorpionsABSTRACT
<p><b>OBJECTIVE</b>To investigate the relation ship of estrogen receptor 2 gene (ESR2) polymorphism associated with intrahepatic cholestasis of pregnancy (ICP) in Chengdu of China.</p><p><b>METHODS</b>By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, the Rsa I polymorphism in exon 5 and the Alu I polymorphism in exon 8 of ESR2 were detected in 100 pregnant women with ICP (ICP group) and 100 normal pregnant women (control group) in Chengdu.</p><p><b>RESULTS</b>(1) The frequency of the allele A of Alu I polymorphism in exon 8 was significantly higher in ICP group than in control group (P=0.031, OR=1.975), so did the frequency of the Aa+AA genotypes (P=0.028, OR=2.144). (2) The genotype distributions (rr, Rr and RR) and allele frequencies (r and R) of Rsa I polymorphism in exon 5 were not significantly different between the two groups (P>0.05).</p><p><b>CONCLUSION</b>The Alu I polymorphism in exon 8 of ESR2 may be associated with the susceptibility of ICP in Chengdu. The Aa+AA genotype significantly elevated the risk suffering from the ICP. The Rsa I polymorphism in exon 5 of ESR2 is not associated with the risk getting the ICP in Chengdu.</p>