OBJECTIVE: To observe the effects of rhubarb aglycone on pathological changes and activity of matrix metalloproteinase in cerebral ischemic tissue in rats with bone marrow mesenchymal stem cell (BMSC) transplantation, and to explore the action mechanisms of rhubarb aglycone in protecting against brain micrangium injury in rats. METHODS: The BMSCs were purified and amplified by methods of adherence and selection in vitro. One hundred and ninety rats were randomly divided into sham-operated group, untreated group, rhubarb aglycone group, BMSC transplantation group (abbreviated as transplantation group) and BMSCs combined with rhubarb aglycone group (abbreviated as combination group). Middle cerebral artery occlusion (MCAO) model was duplicated with nylon thread. Rats of transplantation and combination group were transplanted with BMSCs via carotid artery after 24-hour reperfusion. Rhubarb aglycone was used by intragastric administration in the rhubarb aglycone group and the combination group. The brain samples were taken at 7, 14 and 28 days after transplantation. Brain micrangium pathological changes were observed by light microscope, and immunohistochemical method was used to determine the expressions of immunoglobulin G (IgG), type IV collagen (Col IV), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Comparison with the normal control group revealed that brain micrangium in rats in the untreated group was obviously mutilated and damaged, the expression of IgG and MMP-9 increased, and showed a progressively enhanced tendency following the prolongation of reperfusion, while the expressions of Col IV and TIMP-1 were decreased, and TIMP-1 showed a attenuated tendency following the prolongation of reperfusion. Comparing with the untreated group, the improvements of brain micrangium structure in the rhubarb aglycone group (7 days after transplantation), the transplantation group (14 and 28 days after transplantation) and the combination group were significant; expression of IgG and activity of MMP-9 were decreased, while expressions of Col IV and TIMP-1 were increased in the rhubarb aglycone group and the combination group at each time point. The brain micrangium was integral and the expression of Col IV was enhanced in combination group (7 days after transplantation) as compared with those in transplantation group. MMP-9 activity in combination group (14 days after transplantation) was lower than that in the rhubarb aglycone group (14 days after transplantation), while expression of TIMP-1 in combination group also increased significantly as compared with that in transplantation group (28 days after transplantation). CONCLUSION: Rhubarb aglycone can decrease the degradation of basal lamina Col IV and the permeability of brain micrangium in cerebral ischemic rats with BMSC transplantation, and its mechanisms may be related to regulating the balance of MMP-9, especially by increasing the expression of TIMP-1.

OBJECTIVE: To study the protective effects of Naomaitong, a compound traditional Chinese herbal medicine, used together with thrombolysis therapy, on injuries of the lung and stomach in rats with cerebral ischemia. METHODS: Rats were randomly divided into sham-operated group, untreated group, urokinase group (thrombolysis group), Naomaitong group, thrombolysis plus Naomaitong group. Cerebral ischemia was induced in rats by autonomous blood blot and inserted nylon thread. Rats were administrated with thrombolysis therapy through artery 3, 6 and 9 h after cerebral ischemia respectively. Twenty-four hours after the administration, mortality of the rats, and the brain and stomach hemorrhage ratios, as well as the pathological changes of the brain, lung and stomach were observed, and then cerebral infarct size (CIS) and lung water ratio (LWR) were measured. RESULTS: Compared with the sham-operated group, the rat mortality, and the brain and stomach hemorrhage ratios increased, the CIS enlarged, pathological changes of the brain, lung and stomach appeared obvious, and the LWR increased. Naomaitong plus thrombolysis treatment improved the changes above significantly. In the untreated rats with cerebral ischemia, injuries of the brain, lung and stomach were aggravated, the CIS enlarged and the LWR increased in the 9 h group as compared with those in the 3 h group. In the thrombolysis plus Naomaitong group, the pathological changes were improved, the CIS diminished and the LWR reduced. CONCLUSIONS: Injuries of the lung and stomach can be caused by cerebral ischemia, and the impairment was exacerbated following the prolongation of the ischemia. Thrombolysis therapy can cause brain and stomach hemorrhage. Thrombolysis therapy used early can perform protection against the injuries. Naomaitong, used together with thrombolysis, can reduce the mortality, the brain and stomach hemorrhage ratios, and perform protective effects on the injuries of cerebral ischemia.

The glutamate dehydrogenase (EC.1.4.1.4) gene which amplified from the genome of Brevibacterium flavum GDK-9 by polymerase chain reaction was linked with pUCm-T for sequence alignment. Analysis of gdh sequences revealed that the whole sequence is 1927 bp, only one ORF existed, which used ATG as the initiation codon and coded a peptide of 448 amino acids with a calculated molecular weight of 48 kD. The comparability between the cloned gdh sequence to the reported sequence is high to 99.55%. Only the 1190th base mutation (C→A) lead to the change of amino acid sequence (Thr→Asn), the others are not. The recombinant plasmid pXG was then transformed into E. coli XL-Blue and Brevibacterium flavum GDK-9 which was induced by IPTG. SDS-PAGE analysis revealed that there was a clear induced protein band with molecular mass of 48.7 kD on expected position. Standard glutamate fermentations indicated that although the level of GDH increases the intracellular glutamate pool, the level of GDH has no influence on glutamate secretion.

[Summary] All patients with cholesterol side-chain cleavage enzyme ( P450scc) deficiency that have been reported presented with early adrenal failure. Here we described a 35-year-old male presented with infertility as the only initial presenting complaint. He had received two separate surgeries to remove bilateral testicular masses. We reevaluated the resected tumors and found testicular adrenal rest tumor ( TART) pathology in the resected tumor. We profiled steroid hormones and found significantly elevated ACTH. CT scan revealed bilateral adrenal hyperplasia. Mutation screening identified compound heterozygous mutations (R353W and P432L) in the P450scc encoding gene (CYP11A1). The patient was finally diagnosed as congenital adrenal hyperplasia.

Objective To evaluate the value of endobronchial ultrasound elastography in diagnosis of mediastinal lymph nodes by measuring the parameters of endobronchial ultrasound elastography. Methods Between 2016 and 2017, 46 patients with 63 lesions underwent EBUS-TBNA examination were included. Conventional ultrasound and ultrasound elastography were performed respectively to examine 63 lymph nodes before aspiration. Then various features of conventional ultrasound and parameters of ultrasound elastography were analyzed and recorded. Then evaluate the value of ultrasound elastography by pathological results and three-month follow-up data. Result The diagnostic value of ultrasound elastography image type, elasticity score, strain ratio and blue area ratio for malignant lymph nodes were higher than that of conventional ultrasound features in accuracy, sensitivity and specificity (P < 0.05); The accuracy of the boundary was the highest in the diagnosis of malignant lymph nodes. In the ultrasound elastography parameters, the accuracy of the elasticity score was the highest, and the blue area ratio had the largest area under the curve. When the boundary was used as the joint index to judge malignant lymph nodes with elasticity score and area ratio respectively, significantly higher than the diagnostic accuracy of conventional ultrasound and ultrasound elastography alone (P < 0.05). Conclusion The value of ultrasound elastography parameter in mediastinal lymph nodes diagnosis is higher than the conventional ultrasound features and a combination of both applications can improve discrimination accuracy of benign and malignant lymph nodes.

Objective To assess the association of synovial cyclic citrullinated peptide(CCP)expression with T helper 17(Th17) cells/Regulatory T cells (Treg) imbalance,the histological and clinical features of synovitis in rheumatoid arthritis (RA).Methods CCP expression in synovial specimens from 39 patients with RA and 35 controls was detected by immunohistochemistry assay(IH) using 6×His tagged anti-CCP single chain fragment V (ScFv) antibodies,which were generated by pHEN2 phagemid recombinant antibodies display system.The frequencies of Th17/Treg cells in the peripheral blood mononuclear cells (PBMCs) were determined by flow cytometry (FCM).Th17/Treg cells associated cytokines were analyzed by enzyme-linked immunosorbent assay (ELISA).The histological scores and clinical features of synovitis were included in the study.Chi-square test and ANOVA were used for statistical analysis.Results ① The prevalences of synovial C CP expression were significantly different between RA group and the control(76.9％ and 11.4％ respectively,X2=31.9,P＜0.01).② The frequencies of Th17 cells,Th17/Treg ratio,Th17 cells associated cytokines as IL-6,IL-17a,IL-23,TNF-α,and the Treg cells associated cytokines TGF-31,the serum and synovial fluid anti-CCP antibodies in the RA patients with synovial CCP positive expression were significantly higher in RA patients with CCP positive than those with CCP negative.Disease activity score DAS28 index and histological features quantified variations of the synovial biopsy specimens (synoviocyte hyperplasia,focal aggregates of lymphocytes,and diffuse infiltrat(e)s of lymphocytes) in RA were higher in synovial CCP positive expression patients than in the negative.Conclusion Synovial CCP expression is strongly associated with the Th17/Treg imbalance and synovitis,which may play a crucial role in the pathogenesis of RA.

OBJECTIVETo study the dynamic changes of levels of hepatocyte growth factor (HGF) in tears and their association with corneal haze in rabbits early after epipolis laser in situ keratomileusis (Epi-LASIK).

METHODSTwenty-four New Zealand rabbits received Epi-LASIK with an ablation depth of 100 µm in one eye and of 150 µm in the other eye. Before and at 3, 7, 14, and 30 days after the surgery, the level of HGF in tears collected from the rabbits was measured using enzyme-linked immunosorbent assay (ELISA), and corneal haze was graded after surgery.

RESULTSIn all the rabbits, corneal epithelium healing occurred in 3 to 5 days after Epi-LASIK. Corneal haze appeared 3 days postoperatively in the rabbits accompanied by increased levels of HGF in tears. At 3, 7, 14, and 30 days after the surgery, the rabbits with an ablation depth of 150 µm showed more obvious corneal haze (P<0.05) and significantly higher levels of HGF in tears than those with an ablation depth of 100 µm (P<0.05).

CONCLUSIONIn rabbits receiving Epi-LASIK, HGF levels in tears and the grade of corneal haze show a positive correlation early after the surgery and are both related with the depth of ablation.

OBJECTIVETo construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates.

METHODSThe DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates.

RESULTSTwo small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates.

CONCLUSIONSTwo GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

ADAM Proteins ; blood ; genetics ; ADAMTS13 Protein ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Glutathione Transferase ; metabolism ; Humans ; Male ; Purpura, Thrombotic Thrombocytopenic ; blood ; genetics ; metabolism ; von Willebrand Factor ; genetics ; metabolism

Objective To evaluate the efficacy of Epipolis laser in situ keratomileusis(Epi-LASIK)and laser in situ keratomileu- sis(LASIK)on high myopia patients.Design Prospective,case-controlled study.Participants 62 patients(123 eyes)with high myopia. Methods 62 patients(123 eyes)underwent Epi-LASIK or LASIK surgery for high myopia:28 patients(56 eyes)underwent Epi-LASIK and 34 patients(67 eyes)underwent LASIK.The differences in postoperative uncorrected visual acuity(UCVA),best corrected visual acuity(BCVA),refraction,and root mean square(RMS)of high-range wavefront aberration were compared one-week,one-month, three-months and six-months postoperatively.Main Outcome Measures UCVA,BCVA,refraction,and RMS of high-range wavefront aberration.Results There was no serious complication during and after the operation.The recovery of postoperative UCVA after Epi-LASIK was slower than that of LASIK.One week postoperatively,the proportion of UCVA≥0.8 of Epi-LASIK(46.4%)was less than that of LASIK(77.6%)(P=0.0003).No significant differences were found in those of Epi-LASIK(85.7%,94.6%,91.1%)and those of LASIK(92.5%,95.5%,94.0%)one-month,three-months and six-months postoperatively(P=0.590,0.822,0.530).BCVA was same after Epi-LASIK and LASIK.The proportion of mean spherical equivalents within?0.50D for Epi-LASIK(42.9%,51.8%,60.7%,64.3%)had no difference with those for LASIK(53.7%,59.7%,71.6%,73.1%)one-week,one-month,three-months and six-months postoperatively (P=0.230,0.378,0.200,0.290).The postoperative RMS increased after both surgeries,especially after LASIK.At postoperative one-month,three-months and six-months RMS of Epi-LASIK(1.51?0.77)?m,(1.32?0.76)?m,(1.18?0.71)?m were much higher than the (0.87?0.27)?m preoperative ones(P=0.016,0.019,0.026).At postoperative one-month,three-months and six-months RMS of LASIK (2.41?0.81)?m,(2.17?0.63)?m,(1.89?0.87)?m were also much higher than the preoperative(0.91?0.22)?m(P=0.011,0.008,0.006). There were significant differences between the RMS of Epi-LASIK and LASIK one-month,three-months and six-months postoperatively (P=0.039,0.035,0.033).The I grade haze was found in two eyes after Epi-LASIK.Conclusion Epi-LASIK has better visual quality re- sult than LASIK on correcting high myopia.(Ophthalmol CHN,2007,16:336-339)

Objective To calculate the electric field intensity and transmembrane voltage of spherical cells and ellipsoi -dal red blood cells ( RBC) in an extremely low frequency electromagnetic field .Through this calculation , we can provide reference to the search for interaction targets and mechanics between the extremely low frequency electromagnetic field and organisms.Methods The Finite Element Method was used in the numerical computation for the spherical cell model and the ellipsoidal RBC model .Results The electric field intensity of the two types of cells on the cellular membrane was both significantly higher than the applied electric field strength , and the values of the induced field strength and transmembrane voltage varied with the direction of the electric field periodically .Conclusion The cell shape and direction of the applied electric field are not the main determinants of the cellular membrane electric field intensity and the transmembrane voltage compared with electromagnetic parameters .The distribution of the electric field intensity and transmembrane voltage are re-lated to the direction of the applied electric field.