?AlM: To observe the effect of Qingguang'an on elastic fiber, MMP-7, TlMP-1 in scarring area of filtration canal after glaucoma surgery through the four Qingguang'an effective groups and Qingguang'an granules, to discuss and compare their mechanism of action on scarring area of filtration canal.?METHODS:Four effective components of Qingguang'an were used in groups D, E, F, G and H after glaucoma surgery, compared with group A ( blank ) , group B (model) and group C ( MMC) to observe the effect of elastic fiber, MMP-7, TlMP-1 in scarring filtration canal.?RESULTS:Compared with the preoperative basic lOP and 2d , 1, 2, 4wk postoperative lOP of groups C, E and H, the lOP of three group rose up slower than other groups, and kept the lowest data at 28d. There was significant difference compared with the rest of A, B, D, F, G groups (P<0. 05). The area and density of elastic fiber in surgery group were significantly different with that of black control group ( P<0. 05 ), but there were no statistical differences between groups C and H, groups C and F, groups H and E (P>0. 05). The difference was statistically significant among other groups (P<0. 01).?CONCLUSlON:The scarring area of filtration canal after glaucoma surgery is the major reason which lead to the failure of surgery. Qingguang'an effective group 2, Qingguang'an granules and MMC could reduced the scar tissue by restrained the elastic fiber, TlMP - 1 and increased the MMP-7. By observing the experimental results that both Qingguang'an effective group 2 and Qingguang'an granules could restrained the scarring area of filtration canal, the effects were unbiased, Qingguang'an granules group is better than effective group 2.

Objective To investigate the effects of Longdan Xiegan Decoction on the expression of Notch signaling-related genes in rats with experimental autoimmune uveitis (EAU).Methods Totally 24 Lewis rats were randomly divided into normal control group,EAU model group and Longdan Xiegan Decoction treatment group.A rat model of EAU was established in the EAU model group and Longdan Xiegan Decoction treatment group,followed by intragastrical administration of Longdan Xiegan Decoction in the latter group after successful modeling.On day 12 after different treatments,the spleen and lymph nodes were isolated from rats of all the three groups for the collection of CD4 + T ceils,and the levels of both Th17 and Treg cells were analyzed by flow cytometry to calculate the ratio of Th17 to Treg cells.Meanwhile the levels of Notch1,Notch2,Notch3,Notch4 mRNAs expression were monitored by qRT-PCR.Moreover,the expressions of Notch signaling-related proteins were further detected using ELISA technique.Results Flow cytometry showed increased Th17 cell level but decreased Treg cell level in the EAU model group when compared with the normal control group.However,after treatment with Longdan Xiegan Decoction,the Th17 level was decreased,whereas the Treg level was increased,and the ratio of Th17 to Treg gradually restored to equilibrium when compared to EAU model group.qRT-PCR results showed that the expression levels of Notch1,Notch2 and Notch4 mRNAs in Longdan Xiegan Decoction treatment group were significantly higher than those in the normal control group (all P ＜ 0.01),but lower than those in EAU model group on day 12 after treatment (all P ＜ 0.01),without Notch 3 expression in the spleen and lymph nodes.In addition,Notch 1,Notch 2 and Notch 4 protein levels of the spleen and lymph nodes in Longdan Xiegan Decoction treatment group showed the similar tendency as compared to those of Notch 1,Notch 2 and Notch 4 mRNAs (all P ＜ 0.01),and the expression of Notch 3 protein was still not observed.Conclusion Longdan Xiegan Decoction can effectively relieve the imbalance of Th17/Treg cells in EAU rats,and its mechanism may involve the differentiation of naive CD4+ T cells into Th17 and Treg cells,which is regulated by Notch signaling.

Objective To investigate the regulatory roles of rno-miR-30b-5p in the expression of Atg5,Atg12 and Becn1 autophagy genes and their expressions in rats with experimental autoimmune uveitis (EAU).Methods Application of dual luciferase report system was conducted to detect the regulatory roles of rno-miR-30b-5p in Atg5,Atg12 and Becn1 gene expression.Lewis rats were randomly divided into control group and EAU group,with 6 rats in each group.Next,rats in EAU group were immunized to establish EAU model.After treatments,Genesis-D fundus camera was used to observe the fundus inflammation every day.After immunization for 12 days,the pathological features of rat ciliary body and retina were detected,and meanwhile,the spleen and lymph nodes in both groups were isolated to detect the expressions of rno-miR-30b-5p,Atg5,Atg12 and Becn1 genes by quantitative PCR (Q-PCR);the levels of autophagy related proteins were determined by ELISA.Results Dual luciferase report gene expression assay confirmed that Atg5,Atg12 and Becn1 were target genes of rno-miR-30b-5p.Twelve days after immunization,compared with the control group,rats in the EAU group had severe iris adhesions and severe blood vessel swelling,and pathological examination revealed massive infiltration of inflammatory cells in the ciliary body and retina.Furthermore,rno-miR-30b-5p mRNA was 0.46 ±0.01,0.29 ±0.17in the spleen and lymph nodes in EAU group,respectively,which was down-regulated when compared with the control group (P ＜0.01);whereas the expressions of Atg5,Atg12 and Becn1 were significantly upregulated,and the differences were statistically significant (all P ＜ 0.05).ELISA results showed that Atg5,Atg 12 and Becn 1 protein expression levels in the spleen and lymph nodes in EAU rats were significantly higher than those in the control group (all P ＜ 0.05).Conclusion rno-miR-30b-5p can regulate the expressions of Atg5,Atg12 and Becn1 autophagy-related genes.The down-regulation of rno-miR-30b-5p expression in the spleen and lymph nodes in EAU rats can significantly up-regulate the expressions of Atg5,Atg12 and Becn1 genes,thereby regulating the pathogenesis of uveitis.

OBJECTIVETo obtain large amount of differentiated chondrocytes in vitro, examine and compare the biological characterization of rabbits' articular chondrocyte cultured in different density in vitvo.

METHODSFrom November 2001 to June 2004, articulate tissues were obtained from the joints of the adult rabbits. Chondrocytes were isolated from the cartilage tissue with type II collagenase digestion and cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS). The chondrocytes were cultured with low density of monolayer culture and high density of confluent culture respectively. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry.

RESULTSWhen chondrocytes cultured in monolayer and in low density, it proliferated rapidly during the three generations, but with the same time, dedifferentiation was also rapid. After the third passage, most of the passage cells lost the phenotype, and the proliferation also stagnated. While chondrocytes cultured in high density, dedifferentiation slowed down. And even the phenotypes of the dedifferentiated chondrocyte which were cultured in low density could reduced partly by followed high density culture.

CONCLUSIONSCulture chondrocytes by high density in vitro can effectively maintain the differentiated phenotype of chondrocyte. It also keeps the proliferation character as monolayer culture. The dedifferentiated chondrocyte caused by many passages could redifferentiate partly. So it is indicated that confluent culture of original or expanded chondrocytes in high density is a better culture methods than culture in low density.

Animals ; Cartilage, Articular ; cytology ; Cell Culture Techniques ; methods ; Cells, Cultured ; Chondrocytes ; cytology ; Female ; Male ; Rabbits

OBJECTIVETo investigate the differential expression of different types of Smads in keloids, normal scars and normal skins and its possible clinicopathological significance.

METHODSRT-PCR and Western blot methods were used to examine the expression of Smads mRNA and proteins level in 10 cases of keloid, in 10 cases of normal scar and in 10 cases of normal skin tissues and fibroblasts. Fibroblasts of keloid, normal scar and normal skin were cultured in vitro. The expression difference were compared and analyzed by t-test, there was statistical difference when P < 0.05.

RESULTSThe mRNA and protein expression of inhibitory Smad7 were significantly down regulated in keloid compared with normal scar (P < 0.05) and normal skin (P < 0.05). However, no significant difference of the mRNA and protein expression of Smad2, 3 and the protein expression of phosphorylation of Smad2, 3 in keloid, normal scar, normal skin tissues and fibroblasts.

CONCLUSIONSThe decreased expression of Smad7 in keloid might play a significant role in the increased TGF-beta1/Smads signal transduction, which can not be terminated by autologous negative feedback cycle.

Adolescent ; Adult ; Female ; Humans ; Keloid ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Signal Transduction ; Smad Proteins ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Young Adult

Ischemia-reperfusion injury (IRI) has been recognized as a serious problem for therapy of cardiovascular diseases. Calcium regulation appears to be an important issue in the study of IRI. This article reviews calcium regulation in myocardial and vascular IRI, including the calcium overload and calcium sensitivity in IRI. This review is focused on the key players in Ca(2+) handling in IRI, including membrane damage resulting in increase in Ca(2+) influx, reverse-mode of Na(+)-Ca(2+) exchangers leading to increased Ca(2+) entry, the decreased activity of sarcoplasmic reticulum (SR) Ca(2+)-ATPase causing SR Ca(2+) uptake dysfunction, and increased activity of Rho kinase. These key players in Ca(2+) homeostasis will provide promising strategies and potential targets for therapy of cardiovascular IRI.
Animals ; Calcium ; metabolism ; Heart ; physiopathology ; Homeostasis ; Humans ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism

Hypertension is a common cardiovascular disease and can induce many complications, such as stroke and coronary heart disease. The purpose of the present study was to investigate the effect of ischemia/hypoxia on mesenteric artery vasomotor function in spontaneously hypertensive rats (SHR). Rat mesenteric arterial rings were cultured in modified ischemia-mimetic solution in a hypoxia incubator for a certain time period. Isometric tension changes of isolated mesenteric arterial rings were recorded continuously by a myograph system. The results obtained were as follows: In SHR group, the maximum contractions to KCl and phenylephrine (PE) were increased, and the maximum relaxation to acetylcholine (ACh) was decreased, compared to those in Wistar-Kyoto (WKY) rats group. Compared with SHR group and WKY with acute ischemia/hypoxia (WKY+H) group, SHR with acute ischemia/hypoxia (SHR+H) increased the maximum contractions induced by KCl and PE and inhibited the maximum relaxations by ACh. In SHR+H and SHR groups, the vasodilation induced by ACh was unaffected by N(G)-nitro-L-arginine methylester (L-NAME), whereas in WKY group, the relaxation to ACh was attenuated by L-NAME. CaCl2-induced contraction in depolarized rings in SHR+H group significantly shifted to the left compared with SHR group. In Ca(2+)-free K-H solution, the maximum contractions induced by PE and caffeine were increased in SHR+H group compared to those in WKY+H group; the PE- and caffeine-induced contractions were also enhanced in SHR group versus WKY group; the maximum contraction induced by PE was significantly increased in SHR+H group versus SHR group. These findings suggest that acute ischemia/hypoxia aggravates mesenteric artery dysfunction in SHR. The mechanism may be related to the decreased NO generation and increased sarcoplasmic reticulum Ca(2+) release.
Animals ; Calcium ; metabolism ; Endothelium, Vascular ; metabolism ; physiopathology ; Hypertension ; physiopathology ; Hypoxia ; physiopathology ; In Vitro Techniques ; Male ; Mesenteric Arteries ; physiopathology ; Muscle, Smooth, Vascular ; physiopathology ; Nitric Oxide ; biosynthesis ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Vasomotor System ; physiopathology

OBJECTIVETo observe the effect and mechanism of zoledronate on prevention of collapse in an animal model of osteonecrosis.

METHODSIschemic osteonecrosis was surgically induced in 16 SD rats (which were further divided into zoledronate group and placebo group); another 8 rats were used as sham surgery group (n=8). The animals were killed 5 weeks after surgery. Radiographic, Micro-CT, histological, and immunohistochemical assessments were performed.

RESULTSRadiographic assessment showed better preservation of the femoral head shape in the zoledronate group than in the placebo group but not significantly different from the sham surgery group. Micro-CT assessment showed higher total volume, bone volume, and total mineralized content in the zoledronate group(all P0.05). Compared with the placebo group, the zoledronate group had reduced osteoclast and osteoblast activity, as confirmed by histological examinations.

CONCLUSIONZoledronate can decrease the femoral head deformity by reducing the osteoclast activity while suppressing new bone and vessels formation in a rat model of traumatic osteonecrosis, and therefore may delay the collapse of femoral head.

Animals ; Diphosphonates ; therapeutic use ; Disease Models, Animal ; Femur Head ; drug effects ; pathology ; Femur Head Necrosis ; drug therapy ; pathology ; Imidazoles ; therapeutic use ; Male ; Osteoblasts ; drug effects ; pathology ; Osteoclasts ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.
Animals ; Bacillus thuringiensis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; pharmacology ; Cloning, Molecular ; Endotoxins ; biosynthesis ; genetics ; pharmacology ; Hemolysin Proteins ; biosynthesis ; genetics ; pharmacology ; Microscopy, Atomic Force ; Moths ; Plasmids ; Recombinant Proteins ; biosynthesis ; pharmacology