BACKGROUND:Research has been proved that Fasudil,a Rho kinase inhibitor,can effectively inhibit the onset of secondary spinal cord injury.OBJECTIVE:To investigate the synergistic effect of bone marrow masenchymal stem cell (BMSC) transplantion and Fasudil on motor functional recovery following spinal cord injury.DESIGN,TIME AND SETTING:A randomized controlled animal experimant was performed at institute of Endocrinology,Tianjin Medical University from November 2008 to March 2009.MATERIALS:A 1-month-old SD rat was obtained to extract BMSCs.Another 30 healthy female SD rats were used to establish spinal cord injury models,and they were then randomly divided into single injury group,cell transplantation group,and cell transplantation + Fasudil group,with 10 rats for each group.Fasudil was provided by Tianjin Hongri Pharmaceutical Co.,Ltd.METHODS:One week after modeling,spinal cord injury was exposed in the cell transplantation group and cell transplantation+Fasudil group,and 10 μL BMSC suspension was inserted into the injured region.Otherwise,6 hours later rats in the cell transplantation +Fasudil group were treated with an intraperitoneal injection of 10 mg/kg Fasudil,twice a day for one successive week.MAIN OUTCOME MEASURES:Hindlimb motor function was detected using inclined plane test.The Phosphorylated-ERM protein expression was detected by hernatoxylin-eosin staining,pathology and horseradish peroxidase (HRP) nerve trace,and Western blot.RESULTS:Eight weeks after modeling,degree of inclined plane was significantly increased in cell transplantation group and cell transplantation + Fasudil group compared with single injury group (P ＜ 0.05,P ＜ 0.01);while the increased value in the cell transplantation group was significantly greater than cell transplantation + Fasudil group (P ＜ 0.05).Broken myeloid tissue and cavitation were observed in the single injury group;a few of neuraxis-like structures were observed in the cell transplantation group and cell transplantation + Fasudil group,but the cavity in the cell transplantation group was larger than cell transplantation + Fasudil group.HRP-pesitive nerve fibers were detected at T_8 segment or even above in the single injury group and increased in cell transplantation group,in particular in cell transplantation + Fasudil group.Phospho-ERM protein expression in the single injury group and cell transplantation group was significantly greater than cell transplantation + Fasudil group (P ＜ 0.05).CONCLUSION:BMSC transplantation can promote hindlimb motor functional recovery following spinal cord injury,while the combined application of cell transplantation and Fasudil may cause a synergistic effect.

Objective To study dynamic changes of gene expressions and protein synthesis of Runx2 (runt-related transcription factor-2), Osterix, osteocalcin and AJ18 inside the femoral head with steroid-induced osteonecrosis after mechanical stress stimulation in rats. Methods A total of 50 Wistar rats (half male in sex) weighing 250-270 g (mean 260 g) were involved in this study and randomly divided into experimental group (40 rats) and normal group (10 rats). The rats in experimental group were injected with dexamethasone (20 mg/kg) via bilateral gluteus maximus alternatively once a week and then trained on laboratory animal treadmill twice weekly to make rat model of femoral head necrosis. After identifying the successfully induced model by Hematoxylin and eosin stain, glucocorticoid injection was ceased and the experimental group was randomly divided into model control group, 4 weeks, 6 weeks, 8 weeks groups after hormone training stopped. Then, total RNA and total protein were extracted from femoral head for detecting dynamic changes of genes expressions and proteins synthesis of Runx2, Osterix, osteocalcin and A J18 after mechanical stress stimulation inside the femoral head with steroid-induced osteonecrosis by means of real-time quantitative PCR and Western blot assay. Results In 4 weeks, 6 weeks, 8 weeks groups after hormone training stopped, the gene expressions and proteins synthesis of Runx2, Osterix and osteocalein were reduced more significantly compared with model control group, mBNA expression values of Runx2, Osterix and osteoealcin were 0. 1809, 0. 1639, 0. 1374 and 0. 4219, 0. 3026, 0.2652 and 0. 2857, 0.2027, 0. 1583 times of those in model control group. The expressions of Runx2, Osterix and osteocalcin showed a downward trend with time. The mBNA expression and protein synthesis of AJ18 at 4th, 6th and 8th weeks after hormone training stopped were 2.6391,4. 2718 and 5. 3165 times of model control group. Conclusions In addition to hormonal factors, inappropriate mechanical stress inhibits expressions and proteins synthesis of Runx2, Osterix and osteocalcin, while the expression and protein synthesis of AJ18 are upgraded in early steroid-induced femoral head necrosis in rats.

ObjectiveTo observe the effect of telmisartan on plasma levels of inflammatory cytokine in coronary artery disease (CAD) patients complicated with diabetes mellitus and hypertension after percutaneous coronary intervention.MethodsFifty CAD patients who just had undertook angioplasty and implanted stents were randomly divided into two groups, the test group (telmisartan group, n=25) and control group (perindopril group, n=25). After treatment, patients were followed-up for 6 months; plasma samples were collected from each patient before and after percutaneous coronary intervention. Then plasma levels of C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunoassay. The changes of cholesterol, fasting plasma glucose (FPG), insulin and insulin resistance index (IRI) were observed.ResultsAt the end of 6 months, plasma levels of CRP and MCP-1 of patients in the test group significantly declined (P<0.01), and showing a inversely correlation with FPG (P<0.01), and FPG, insulin and IRI also declined. In the control group, only CRP and MCP-1 declined (P<0.05). Meanwhile, the frequency of cardiovascular events in test group was significantly lower than that in the control group.ConclusionTelmisartan can decline plasma levels of CRP, MCP-1 and frequency of cardiovascular events as well as increasing insulin sensitivity and improving glucose metabolism to unstable angina patients.

Objectives:To establish hybridomas secreting monoclonal antibodies(McAbs) against O6-methylguanine-DNA methyltransferase(MGMT) and to observe the relationship between MGMT expression and clinical responses to ACNU and BCNU in human brain tumors.Methods:The hybridomas were established by cell fusion.MGMT expression in 60 glioma specimens was detected by means of immunohistochemical assay.Results: Seven hybridomas secreting McAbs against MGMT were obtained.Thirty tumor specimens had no detectable or low level of MGMT expression(Mer-), while 30 specimens had high level of MGMT expression(Mer+). The Mer- patients showed more sensitive to ACNU and BCNU than the Mer+ patients.Conclusions: The high specific hybridomas secreting monoclonal antibodies(McAbs) against MGMT were established.The preliminary study indicated that MGMT negative tumors were sensitive to ACNU and BCNU, whereas MGMT positive ones were more resistant to nitrosourea drugs.

OBJECTIVETo explore the strategies which reduce the amount of xenoantigen Galalpha1,3Gal.

METHODSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human alpha-galactosidase transgenic mice were produced. The Galalpha1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed.

RESULTSHuman alpha-galactosidase gene alone reduced 78% of Galalpha1,3Gal on PEDSV.15 cell surface while human alpha-galactosidase combined with alpha1,2-fucosyltransferase genes removed Galalpha1,3Gal completely. Decrease of Galalpha1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of alpha-galactosidase gene and alpha1,2-fucosyltransferase gene. RT-PCR indicated positive human alpha-galactosidase gene expression in all organs of positive human alpha-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galalpha1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice.

CONCLUSIONSHuman alpha-galactosidase gene and alpha1,2-fucosyltransferase gene effectively reduce the expression of Galalpha1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human alpha-galactosidase in mice can also eliminate the Galalpha1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.

Animals ; Antigens, Heterophile ; metabolism ; Cell Death ; Cells, Cultured ; Disaccharides ; metabolism ; Endothelial Cells ; metabolism ; Fucosyltransferases ; genetics ; metabolism ; Graft Rejection ; genetics ; Humans ; Mice ; Mice, Transgenic ; Spleen ; cytology ; Swine ; Transfection ; alpha-Galactosidase ; genetics ; metabolism

Objective:To evaluate the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in striatum in paclitaxel-induced neuropathic pain and the relationship with GABA B receptors in rats. Methods:Healthy clean-grade male Sprague-Dawley rats, aged 6-9 weeks, weighing 180-220 g, were used in this study.The neuropathic pain model was established by intraperitoneal injection of paclitaxel 2 mg/kg once every 2 days for 7 consecutive days in anesthetized rats, and then intrathecal catheterization was performed.Fifty rats in which intrathecal catheters were successfully implanted were divided into 5 groups ( n=10 each) using a random number table method: paclitaxel group (P group), paclitaxel plus normal saline group(N group), paclitaxel plus lentivirus empty vector group (BV group), paclitaxel plus HCN4 channel lentivirus group (H group), and paclitaxel plus HCN channel inhibitor ZD7288 group (I group). Ten rats of the same age were selected as the blank control group (C group). At 15 days after intraperitoneal injection of paclitaxel, normal saline 20 μl was intrathecally injected in group N, group BV received intrathecal injection of HCN4 channel lentivirus empty vector 1×10 8 TU/ml, 20 μl, group H received intrathecal injection of HCN4 channel lentivirus 1×10 8 TU/ml, 20 μl, and group I received intrathecal injection of ZD728830 μg, 20 μl.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before paclitaxel injection and 14 and 21 days after injection.The cerebral striatum tissues were obtained at T 2, and the expression of HCN4 channel and GABA B receptors was determined by immunohistochemistry and Western blot. Results:Compared with group C, the MWT was significantly decreased, HCN4 channel expression was up-regulated, and GABA B receptor expression was down-regulated in group P ( P<0.05). Compared with group P, the MWT was significantly increased, HCN4 channel expression was down-regulated, and GABA B receptor expression was up-regulated in group H and group I ( P<0.05), and no significant change was found in the parameters mentioned above in group N and group BV ( P>0.05). Conclusion:Up-regulation of expression of HCN4 channels in striatum can induce down-regulation of GABA B receptor expression, which is involved in the pathophysiological mechanism of paclitaxel-induced neuropathic pain in rats.

OBJECTIVETo determine the origin of a supernumerary small marker chromosome found in a fetus using prenatal BACs-on-Beads (BoBs) and single nucleotide polymorphism array (SNP-array) assays.

METHODSThe fetal sample was subjected to chromosomal karyotyping and BoBs analysis, and the results were validated with genome-wide scanning using a SNP microarray.

RESULTSThe fetus was found to have a 47,XX,+mar karyotype. BoBs analysis indicated that there was an amplification between 18p11.32 and 18p11.21, which was verified by the SNP-array assay as a 18.3 Mb duplication occurring at 18p11.32q11.1.

CONCLUSIONThe karyotype of the fetus was determined as 47,XX,+der18(18p11.32?18q11.1::18q11.1?18p11.32). The duplication has involved important genes including SMCHD1, LPIN2 and TGIF1, which may result in severe malformations in the fetus.

Adult ; Aneuploidy ; Chromosomes, Artificial, Bacterial ; genetics ; Chromosomes, Human, Pair 18 ; genetics ; Female ; Humans ; Karyotyping ; Microarray Analysis ; methods ; Polymorphism, Single Nucleotide ; Pregnancy ; Prenatal Diagnosis ; methods

Objective    To evaluate the short-term therapeutic effect of extended adventitial inversion with graft eversion anastomosis technique in the root treatment of acute type A aortic dissection (ATAAD). Methods    From November 2019 to July 2020, 28 patients with ATAAD were treated by extended adventitial inversion with graft eversion anastomosis technique in the Department of Cardiovascular Surgery, Dalian Municipal Central Hospital, including 19 males and 9 females, aged 60.11±11.11 years. The intima of the ascending aorta was trimed to 5 mm above the sinotubular junction. The adventitia of the ascending aorta was longitudinally cut to the reserved intima margin along the junction of the three aortic valves. The extended adventitial inversion was sutured continuously, no coronary sinus was sutured over the aortic annulus, and the left and right coronary sinus was sutured above the coronary ostium. The anastomotic graft was everted and inserted into the aortic lumen, and the everted graft was continuously sutured at the level of sinotubular junction which was 5 mm away from the edge of graft. Results    There was no intraoperative death, intractable root hemorrhage, residual root false lumen, root dilatation, anastomotic hematoma or other complications. There was no recurrence of the pain in the back of all patients, and the results of the CT angiography were not significantly changed. In 22 patients with no regurgitation, only 1 (4.55%) patient had a mild regurgitation. In 6 patients with mild aortic regurgitation, the disappearance rate of regurgitation was 50.0% (3/6). Conclusion    The treatment of extended adventitial inversion with graft eversion anastomosis technique in the root treatment of aortic dissection eliminates the residual dissection at the root. The anastomotic hemorrhage is prevented, the root structure of aortic dissection is reconstructed and strengthened, the root function is restored, and the possible expansion of the root is prevented. The short-term results are satisfactory.

While the majority of all human cancers counteract telomere shortening by expressing telomerase, ~15% of all cancers maintain telomere length by a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). Here, we show that high load of intrinsic DNA damage is present in ALT cancer cells, leading to apoptosis stress by activating p53-independent, but JNK/c-Myc-dependent apoptotic pathway. Notably, ALT cells expressing wild-type p53 show much lower apoptosis than p53-deficient ALT cells. Mechanistically, we find that intrinsic DNA damage in ALT cells induces low level of p53 that is insufficient to initiate the transcription of apoptosis-related genes, but is sufficient to stimulate the expression of key components of mTORC2 (mTOR and Rictor), which in turn leads to phosphorylation of AKT. Activated AKT (p-AKT) thereby stimulates downstream anti-apoptotic events. Therefore, p53 and AKT are the key factors that suppress spontaneous apoptosis in ALT cells. Indeed, inhibition of p53 or AKT selectively induces rapid death of ALT cells in vitro, and p53 inhibitor severely suppresses the growth of ALT-cell xenograft tumors in mice. These findings reveal a previously unrecognized function of p53 in anti-apoptosis and identify that the inhibition of p53 or AKT has a potential as therapeutics for specifically targeting ALT cancers.