Relationship between Muscle Mass of Limbs and aGVHD in Patients with Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

OBJECTIVE@#To explore the correlation of limb muscle mass and acute graft-versus-host disease.@*METHODS@#Clinical data from 144 patients treated by allo-HSCT in Guangzhou First People's Hospital were collected and analyzed retrospectively. The age, sex, diagnosis, donor age, sex of the donors, preparative regimen, ATG dose, HLA match, graft source, and number of infused stem cells of the patients were collected as baseline information. Meanwhile, bioelectrical impedance principle (BIA) was used to measure the limb muscle mass, body weight, body mass index (BMI), waist-to-hip ratio, upper arm muscle circumference, triceps skinfold thickness, and body fat rate of the patients before and after transplantation, so as to compare the changes of limb muscle mass and investigate its correlation with aGVHD.@*RESULTS@#It was found that 61.11% of allo-HSCT patients showed muscle mass loss, and the proportion of male and female was 35.42% and 25.69%, respectively. There were reduction in the body weight, BMI, upper arm muscle circumference and muscle mass of limbs after transplantation as compared with those before transplantation (P<0.05). By comparing with the cumulative incidence of aGVHD between the patients in low muscle mass group and normal muscle mass group, it was found that the cumulative incidence of Ⅱ-Ⅳdegree aGVHD in patients with low muscle mass (30.38%) was higher than those with normal muscle mass (8.93%), which showed statistical difference (P<0.05). Univariate analysis showed that muscle mass, the sex of the donors, and preparative regimen were the influencing factors of aGVHD (P<0.05). Binary logistic regression showed that low muscle mass was the independent risk factor affecting aGVHD (P<0.05).@*CONCLUSION@#Patients treated by allo-HSCT shows a decline in muscle mass after transplantation, and the incidence of aGVHD is high in patients with low muscle mass. Therefore, the assessment of muscle quality in early stage in patients with HSCT can facilitate earlier detection of aGVHD.

Effect of 1,25(OH)2D3on type Ⅰ collagen secretion in adipose-derived mesenchymal stem cells and its mechanism / 中国组织工程研究

BACKGROUND: Adipose-derived mesenchymal stem cells (ADMSCs) have been reported to improve wound healing. However, type I collagen secreted by ADMSCs will contribute to scar formation. Therefore, inhibiting type I collagen secretion from ADMSCs will strengthen its clinical application. OBJECTIVE: To investigate the effect of 1,25(OH)2D3on secretion of type I collagen by ADMSCs and its mechanism. METHODS: Human ADMSCs were isolated by collagenase digestion, and identified by flow cytometry. ADMSCs at passage 4 were cultured in DMEM/F12 medium containing different concentrations of 1,25(OH)2D3(10-7, 10-8, 10-9, 10-10and 0 mol/L) respectively for 4 days. Then, the concentration of type I collagen in cell supernatant was measured by ELISA. Real-time PCR and western blot were used to detect the expression of Smad3 at mRNA and protein levels and phosphorylated protein Smad3 level in ADMSCs cultured with and without 1,25(OH)2D3. To analyze the contribution of Smad3 to the effect of 1,25(OH)2D3, Smad3 inhibitor was added to culture medium 30 minutes before adding 1,25(OH)2D3, and type I collagen in cell supernatant was detected by ELISA at 4 days after addition of SMAD3 inhibitor. RESULTS AND CONCLUSION: 1,25(OH)2D3inhibited the secretion of type I collagen by ADMSCs in a dose-dependent manner. The results of real-time PCR and western blot showed that the expression of Smad3 was upregulated by 1,25(OH)2D3, and the results of western blot showed that the phosphorylated Smad3 protein level in ADMSCs was significantly increased by 1,25(OH)2D3. Moreover, the inhibition of type I collagen secretion by 1,25(OH)2D3could be blocked by Smad3 inhibitor. These results indicate that 1,25(OH)2D3can inhibit the secretion of type I collagen from ADMSCs by up-regulating the expression of Smad3.

A Preliminary Study of the Therapeutic Role of Human Early Fetal Aorta-derived Endothelial Progenitor Cells in Inhibiting Carotid Artery Neointimal Hyperplasia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endothelial cell damage is an important pathophysiological step of restenosis after angioplasty and stenting. Cell transplantation has great therapeutic potential for endothelial recovery. We investigated the effect of transplanting endothelial progenitor cells (EPCs) derived from human early fetal aortas in rat injured arteries.</p><p><b>METHODS</b>The carotid arterial endothelium of Sprague-Dawley rats was damaged by dilatation with a 1.5 F balloon catheter, and then EPCs derived from human early fetal aortas (<14 weeks) were injected into the lumen of the injured artery in transplanted rats, with an equal volume of normal saline injected into control rats. Rats were sacrificed at 2 and 4 weeks after treatment and transplanted cells were identified by immunohistochemical staining with anti-human CD31 and anti-human mitochondria antibodies. Arterial cross-sections were analyzed by pathology, immunohistochemistry, and morphometry.</p><p><b>RESULTS</b>Green fluorescence-labeled EPCs could be seen in the endovascular surface of balloon-injured vessels after transplantation. The intimal area and intimal/medial area ratio were significantly smaller in the transplanted group than in the control (P < 0.05) and the residual lumen area was larger (P < 0.05). After EPC transplantation, a complete vascular endothelial layer was formed, which was positive for human von Willebrand factor after immunohistochemical staining, and immunohistochemical staining revealed many CD31- and mitochondria-positive cells in the re-endothelialized endothelium with EPC transplantation but not control treatment.</p><p><b>CONCLUSION</b>EPCs derived from human early fetal aorta were successfully transplanted into injured vessels and might inhibit neointimal hyperplasia after vascular injury.</p>

Modification by wheat germ agglutinin delays the ocular elimination of liposome / 药学学报

The purpose of this study is to explore the feasibility of wheat germ agglutinin (WGA) modified liposome as a vehicle for ophthalmic administration. Liposome loaded with 5-carboxyfluorescein (FAM) was prepared by lipid film hydration method. WGA was thiolated and then conjugated to the surface of the liposome via polyethylene glycol linker to constitute the WGA-modified and FAM-loaded liposome (WGA-LS/FAM). The amount of thiol groups on each WGA molecule was determined, and the bioactivity of WGA was estimated after it was modified to the surface of liposome. The physical and chemical features of the WGA-modified liposome were characterized and the ocular bioadhesive performance was evaluated in rats. The result showed that each thiolated WGA molecule was conjugated with 1.32 thiol groups. WGA-LS/FAM had a mean size of (97.40 +/- 1.39) nm, with a polydispersity index of 0.23 +/- 0.01. The entrapment efficacy of FAM was about (2.95 +/- 0.21)%, and only 4% of FAM leaked out of the liposome in 24 h. Erythrocyte agglutination test indicated that after modification WGA preserved the binding activity to glycoprotein. The in vivo ocular elimination of WGA-LS/FAM fitted first-order kinetics, and the elimination rate was significantly slower than that of the unmodified liposome, demonstrating WGA-modified liposome is bioadhesive and suitable for ophthalmic administration.

The function of bushen qiangdu recipe containing serum in OPG/RANKL pathway of ankylosing spondylitis patients / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the mechanism of Bushen Qiangdu Recipe (BQR) for regulating osteoprotegerin receptor activator for nuclear factor kappa B ligand (OPG/RANKL) pathway in ankylosing spondylitis (AS).</p><p><b>METHODS</b>Thirty active AS inpatients or outpatients were recruited from Department of CM Rheumatology, China-Japan Friendship Hospital from January to May 2009. All patients were treated with BQR for 3 successive months, one dose daily, once in the morning and once in the evening. Besides, 30 healthy volunteers were recruited. The serum of patients and volunteers were collected. The osteoblast cell lines hFOB1. 19 were divided into 3 groups: the pre-treatment group, the post-treatment group, and the healthy volunteer group (as the control group). All cell lines were cultured by corresponding culture medium containing each serum. The supernatant from osteoblast cell lines was collected. The protein content of OPG/RANKL was detected using ELISA, and the protein expression of OPG/RANKL was detected using RT-PCR.</p><p><b>RESULTS</b>Compared with the control group, the OPG content, the mRNA and protein expressions of OPG, and the mRNA and protein expressions of OPG/RANKL all decreased, while the mRNA expression of RANKL increased in the pre-treatment group, showing statistical difference (P<0.05, P<0.01). Compared with the pre-treatment group, the OPG content, the mRNA and protein expressions of OPG significantly increased, and the mRNA and protein expressions of OPG/RANKL increased, while the mRNA expression of RANKL decreased in the post-treatment group, showing statistical difference (P<0.05, P<0.01).</p><p><b>CONCLUSIONS</b>AS patients' serum could directly inhibit the expression of OPG in osteoblasts, promote the expression of RANKL, and down-regulate the OPG/RANKL ratio. BQR containing serum might promote the osteogenesis and inhibit the bone resorption possibly through directly up-regulating the OPG/RANKL ratio in osteoblast, thus inhibiting the differentiation and function of osteoclast.</p>

Poly(arginine)8 enhanced intestinal absorption of insulin-loaded nanoparticles / 药学学报

The purpose of this study is to investigate the feasibility of poly(arginine)8 (R8) modified poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a carrier for the oral delivery of insulin. Insulin-loaded PLGA nanoparticle (INS-NP) was prepared by a double emulsion-solvent evaporation method, and R8 was subsequently conjugated to the surface of the INS-NP via polyethylene glycol bridge (R8-INS-NP). The physical and chemical features of the nanoparticles were characterized, and insulin release was determined in vitro. The pharmacokinetics and pharmacodynamics were evaluated by in situ absorption study with the intestinal loop of rats. The blood glucose level was determined by glucose oxidize method and the serum insulin concentration was determined by radioimmunoassay (RIA). The mean diameter of INS-NP was (179.0 +/- 5.2) nm and the polydispersity index was 0.152 +/- 0.042, while the entrapment efficiency was (29.10 +/- 2.59) %. The in vitro release behavior of insulin showed an initial burst effect followed by a stage of slow release. After administrating 10 U x kg(-1) insulin to rats, R8-INS-NPs decreased the plasma glucose level much lower than INS-NPs, meanwhile, D-form R8 substantially enhanced intestinal absorption of insulin much more than L-form R8. Compared to subcutaneous injection, the relative bioavailabilities of insulin were 0.52%, 4.78%, 8.39%, and the pharmacological bioavailabilities were 2.07%, 3.90%, 8.24%, separately. The R8-modified nanoparticles promoted the intestinal absorption of insulin, which might be a potential approach for oral delivery of peptide, protein and even other hydrophilic macromolecules in the future.

Effects of Bushen qiangdu decoction on MMP-9 and TIMP-1 produced by peripheral blood mononuclear cells in patients with ankylosing spondylitis / 中国骨伤

<p><b>OBJECTIVE</b>In order to investigate the roles of metalloproteinase in inflammatory bone destruction in ankylosing spondylitis (AS), and analyze the mechanism of preventing inflammatory bone destruction of Bushen Qiangdu decoction (BSQDD) in AS cases. Comparisons were made on the expressions of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) by peripheral blood mononuclear cells (PBMC) between AS patients and healthy controls. The effect of BSQDD was investigated on the expression and of MMP-9 and TIMP-1 produced by PBMC in AS patients.</p><p><b>METHODS</b>From March 2005 to March 2006, 30 active AS cases of Kidney-asthenia, Du-cold and blood-stasis syndrome were selected as patients group in the China-Japan Friendship Hospital. There are 27 male patients and 3 female patients. The age range is from 16 to 45, averaging (30.8 +/- 8.8) years. Disease duration is from 0.5 to 10 years. Cases received three-month BSQDD treatment were considered as the treatment group. Twenty healthy persons were included in the control group. Serum and PBMC were separated. The PBMC were stimulated by PHA and PMA, and the supernatant was collected. The mRNA expression of MMP-9 and TIMP-1 in PBMC was analyzed by RT-PCR. The content of MMP-9 and TIMP-1 in serum and culture supernatant of PBMC were detected by ELISA.</p><p><b>RESULTS</b>Compared with health control group, the serum concentration of MMP-9 and TIMP-1 in patients group before treatment increased (P<0.01, P<0.05), but the level of MMP-9 and TIMP-1 in the serum of patients after treatment decreased compared with pre-treatment cases (P<0.05). Furthermore,compared with health control group, PBMC of patients group before treatment expressed higher levels of MMP-9 and TIMP-1 both on transcript level and at protein level (P<0.01, P<0.05), and the expression levels of MMP-9 and TIMP-1 in PBMC in patients after treatment both on transcript level and at protein level was down-regulated compared with pre-treatment (P<0.01, P<0.05).</p><p><b>CONCLUSION</b>PBMC of AS patients had a higher potential capacity for MMP-9 and TIMP-1. BSQDD possibly prevented inflammatory bone destruction of AS through inhibiting production of MMP-9 and TIMP-1 produced by PBMC.</p>

Morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line (LSEC line).</p><p><b>METHODS</b>Immunofluorescence staining and fluorescence microscopy were used to detect the classic endothelial cell markers in LSEC line, and flow cytometry was used to analyze the purity of the human LSEC line. The morphology (including W-P bodies and surface fenestrations) and phagocytotic capacity of the human LSEC line were observed by transmission and scanning electron microscope. The proliferation curve of the human LSEC line was analyzed by MTT assay. The functional differences between the human LSEC line and human primary LSEC in expression of ELAM-1 and ICAM-1, activities of fibrinolysis (PAI-1, t-PA, u-PA), releasing of IL-6 and IL-8 were compared respectively by enzyme linked immunosorbent assay. Comparison of the susceptibility to hypoxia-reoxygenation induced apoptosis between the human LSEC line and human primary LSEC were investigated by TUNEL.</p><p><b>RESULTS</b>The established human LSEC line maintained a high proliferative ability and has been passaged for more than 80 times in the absence of any growth factors. Immunofluorescence staining showed that the human LSEC line could express classic endothelial cell marks including von Willebrand Factor (vWF), and could take up acetylated low-density lipoproteins (Ac-LDL). The purity of the human LSEC line was confirmed over 95% by flow cytometric analysis. The W-P bodies and the phagocytosis of Dynabeads was demonstrated by transmission electron microscope. And fenestrations could be found cellular surface with scanning electron microscopy. When compared with human primary LSEC, the human LSEC line has an equivalent responsiveness to tumor necrosis factor in up-regulation of ELAM-1 and ICAM-1. The human LSEC line can also release PAI-1, t-PA, u-PA but can not release IL-6 and IL-8 to TNF-alpha. In contrast, human primary LSEC could release IL-6. The human LSEC line showed higher susceptibility to hypoxia-reoxygenation-induced apoptosis, and the percentage of apoptotic cells was as high as (38.4 +/- 6.7)%, while (28.6 +/- 4.5)% and (7.8 +/- 1.2)% respectively in primary LSEC and in human umbilical vein endothelial cells.</p><p><b>CONCLUSIONS</b>The established human LSEC line maintains the special phenotypes and the major functional characteristics, and especially maintains the high susceptibility to hypoxia-reoxygenation-induced apoptosis. Therefore it is feasible to use this cell line for the study of liver ischemia-reperfusion injury.</p>

Tumor cell-tumor endothelial cell adhesion mediated by alphavbeta3 and alphavbeta5 molecules / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro.</p><p><b>METHODS</b>The expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture.</p><p><b>RESULTS</b>The expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5.</p><p><b>CONCLUSION</b>alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.</p>

Phenotypic and functional characteristics of endothelial cells derived from human liver cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To analyze the phenotypic and functional characteristics of endothelial (T3A) cells derived from human hepatocellular cell carcinoma.</p><p><b>METHODS</b>Endothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking von Willebrand Factor (vWF), CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypic characteristics of T3A cells were analyzed by fluorescence-activated cell sorter (FACS) and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity was evaluated by 3-(4, 5-dimethythiazolyl) -2, -diphenyl-2H-tetrazolium-bromide (MTT) assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The isolated T3A cells exhibited classic "spindle-shape" morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express alphav3, alphavbeta5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells (LSEC). In response to TNFalpha, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor (TF), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and urine plasminogen activator (u-PA) than LSEC in response to TNFalpha.</p><p><b>CONCLUSION</b>Tumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.</p>

Characteristics of human adrenal microvascular endothelial cells in morphology, phenotypes, and function / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the phenotypic and functional characteristics of human adrenal microvascular endothelial cells (AdrEC).</p><p><b>METHODS</b>AdrEC were isolated and purified from a sample of human adrenal tissue by sub-cell clone method. The cells identified by flow cytometry for classical endothelial markers von Willebrand factor (vWF) and CD31, uptake of Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL), as well as phenotypes. The cell fenestrations were checked by scanning electron microscopy. The expressions of endogenous vascular endothelial growth factor (VEGF) mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The glucocorticoid-induced cytotoxicities in different organs-derived microvascular endothelial cells were compared.</p><p><b>RESULTS</b>Human AdrEC expressed those classical endothelial markers such as vWF, CD31, and uptake of Dil-Ac-LDL. The phenotypic analysis indicated that alpha-1 proteinase inhibitor, tumor necrosis factor receptor p55, and intercellular adhesion molecule-1 were expressed in human AdrEC. Scanning electron microscopy demonstrated that there were many microvilli and fenestrations on cellular surface. RT-PCR and immunocytochemistry showed that there was expression of endogenous VEGF in AdrEC. In response to glucocorticoid-induced cytotoxicity, microvascular endothelial cells (MVEC) derived from human brain were highly susceptible, MVEC derived from human lung and human liver sinusoidal endothelial cells were sub-sensitive, while AdrEC were highly resistant.</p><p><b>CONCLUSION</b>Human AdrEC are specially differentiated and have characteristics that are different from other organ-derived MVEC in phenotypes and functions.</p>