小型猪骨髓间充质干细胞超顺磁氧化铁、增强型绿色荧光蛋白双标记及体外磁共振成像示踪的研究

目的 探讨超顺磁氧化铁(SPIO)和增强型绿色荧光蛋白(EGFP)体外双标记、磁共振成像(MRI)示踪1型糖尿病(T1DM)小型猪骨髓间充质干细胞(BMSCs)的可行性和标记方法.方法 采集T1DM小型猪骨髓,密度梯度离心和贴壁培养法分离BMSCs.选用携带EGFP慢病毒(LV)颗粒转染BMSCs.选用SPIO和左旋多聚赖氨酸(PLL)复合物标记LV-EGFP转染的BMSCs.对SPIO标记细胞进行普鲁士蓝染色、透射电镜观察和体外MRI.结果 LV-EGFP与BMSCs转染复数(MOI)为30∶1转染后96 h荧光表达最强,阳性转染率43.2%.Fe3+标记浓度为25 mg/L BMSCs阳性标记率93.1%.透射电镜检测发现,SPIO粒子密集于BMSCs细胞质,次级溶酶体内多见.MRI显示,Fe3+标记浓度为25 mg/L、1×104/ml细胞浓度标记后3周和6周在快速自旋回波序列(TSE)T1WI、TSET2WI及快速小角度激发成像(FLASH)T2*WI信号强度变化率(△SI)依次为12%、41%、63%和7%、28%、46%,均为后二者大于前者(分别P＜0.01和P＜0.05),提示SPIO该标记浓度和细胞密度标记后3周和6周在T2WI及T2*WI呈现较明显的信号变化.结论 采用体外EGFP和SPIO双重标记的方法,结合MRI和细胞化学染色法可以稳定地对T1DM小型猪BMSCs进行体外示踪.

Abstract：

Objectives To track bone marrow stem cells (BMSCs) labeled by enhanced green fluorescent protein (EGFP) and superparamagnetic iron oxide ( SPIO ) -poly-L-lysine (PLL) compound by MRI in vitro for autotransplantation into pancreas of type 1 diabetes miniature pigs. Methods The BMSCs were isolated by density gradient centrifugation and attachment culture from type 1 diabetes minipigs' bone marrow. Expressional intensity of EGFP in BMSCs transfected lentivirus-EGFP with a multiplicity of infection Different magnetic resonance scanning protocols were carried out on various density BMSCs labeled by different concentration of SPIO in various time-point in vitro. Results When SPIO concentration was 25mg/L (count in Fe3 + ), the positive Fe3+ -labeling rate of BMSCs was 93. 1%. Most of SPIO particles in BMSCs' cytoplasm were observed in secondary lysosomes, but they were not detected in important organelle as cell nucleus. Comparing with gelatin the MRI of BMSCs labeled with SPIO in the condition with 1 ×104/ml cells density and 25 mg/L Fe3+ concentration in vitro, the signal intensity changes (△SI) after BMSCs labeled with SPIO 3 weeks and 6 weeks in TSE T1WI, TSE T2WI and FLASH T2 * WI sequences were 12%, 41%, 63% and 7%, 28%, 46% respectively (P ＜ 0.01 and P ＜ 0.05, respectively).Conclusions The data showed that the porcine BMSCs labeled with SPIO and EGFP could be traced successfully in vitro by MRI in the suitable sequences.     

Clinical features of benign convulsions with mild gastroenteritis in Chinese infants

Background

Benign convulsions occur in infants during the course of mild gastroenteritis. It is now recognized as a distinct clinical entity in many countries. However, its occurrence in China has not yet been widely recognized by Chinese pediatricians.

Methods

A retrospective study was conducted in 48 patients with convulsions between January 1, 2004 and December 31, 2009.

Results

The age of onset of gastroenteritis was between 13 months and 24 months in 34 patients (70.83%). The episodes occurred at a distinct autumn/winter peak (75%). The seizures mostly occurred within the first 5 days of gastroenteritis, especially within the first 3 days, peaking on day 2 (39.58%). Thirty-five patients (72.92%) had clustered seizures in their episodes. Most episodes were symmetric, generalized tonic-clonic (83.33%) and brief (93.75%). The seizures were induced by pain and/or crying in 19 (39.58%) patients. Stool culture was positive for rotavirus in 21 (53.85%) of the 39 patients. Twenty patients (20/41, 48.78%) still had clustered seizures after the administration of a single anticonvulsant. The seizures persisted even after the administration of two combined anticonvulsants in 5 (26.32%) of 19 episodes. All patients exhibited normal psychomotor development.

Conclusions

Benign convulsions with mild gastroenteritis are not rare in China, and rotavirus infection is a major cause.     

A case of leukemia cutis presenting as histiocytoid Sweet’s syndrome

Background Leukemia cutis (LC) represents a skin infiltration by leukemic cells. Clinically, it can mimic a wide variety of dermatoses. Methods We report a case of LC presenting with a Sweet’s syndrome‐like eruption and a histiocytoid Sweet’s syndrome histologic manifestation. Results This case may represent distinct and important cutaneous and histopathologic manifestations of LC. Conclusions We believe that peripheral blood or bone marrow cytologic analysis is necessary in cases of LC to rule out the possibility of histiocytoid Sweet’s syndrome. Additional study is needed to further elucidate the relationship between LC and histiocytoid Sweet’s syndrome.     

基于古方数据挖掘珍稀中药人参的用药特点与配伍规律

目的:通过数据挖掘和关联分析等方法,寻找古方中使用珍稀中药人参的用药特点与配伍规律,以验证并拓宽其临床应用。方法:从中医方剂数据中筛选出用量及功效明确、组成含有人参的方剂2 420首。通过Apriori算法及聚类分析对方剂进行数据挖掘。结果:Apriori关联分析得出,人参-甘草,人参-白术,人参-当归,是最常用的二联用药。人参-白术、甘草,人参-白芍、当归,当归-白芍、人参,是最常用的三联用药。小剂量人参与甘草、白术配伍,大剂量人参与甘草配伍;通过聚类提取出4个因子,提取出的方剂分别具有止痛、温补肾阳、降逆止呕、补气行血的功效。结论:人参用量不宜过大,与人参配伍的中药以调和人参药性、治疗脾胃虚寒、温肾散寒止痛4个方面的作用为主,为临床使用与实验研究提供理论依据与实验思路。     

Sphingosylphosphorylcholine stimulates cellular fibronectin expression through upregulation of IL-6 in cultured human dermal fibroblasts

Sphingosylphosphorylcholine (SPC) has been reported to stimulate wound healing by its potent mitogenic effect. Fibronectin (FN) is a cell-adhesion protein that plays an important role in cell migration and collagen deposition during wound healing. In order to elucidate further the mechanism involved in the accelerated wound healing stimulated by SPC, we studied the role of SPC in FN production in cultured human dermal fibroblasts. We demonstrated that SPC dose- and time-dependently enhanced the expression of FN in human dermal fibroblast at the protein and mRNA levels. IL-6 is known to stimulate the production of FN in fibroblasts. SPC also markedly induced IL-6 production in cultured human dermal fibroblasts in a dose- and time-dependent manner. We also demonstrated that FN mRNA expression in human dermal fibroblasts was upregulated 4 h after IL-6 treatment. Moreover, pretreatment with neutralizing anti-IL-6 antibodies partially blocked the upregulation of FN mRNA expression induced by SPC in human dermal fibroblasts. These results indicate that SPC may stimulate FN synthesis through IL-6 production in cultured human dermal fibroblasts.     

老年冠心病合并慢性肾功能不全患者介入治疗围术期应用不同剂量依诺肝素抗凝的有效性和安全性

目的探讨不同剂量依诺肝素抗凝在老年冠心病合并肾功能不全患者介入治疗围术期的安全有效剂量。方法分析2009年1月至2012年12月在我科住院的108例冠心病合并肾功能不全介入治疗围术期应用依诺肝素的患者,按依诺肝素应用剂量共分为3组。A组（n=31）：依诺肝素40mg,皮下注射,每12小时1次。B组（n=35）：依诺肝素40mg,皮下注射,每日1次。C组（n=42）：依诺肝素40mg,皮下注射,每早1次以及依诺肝素20mg,皮下注射,每晚1次。观察各组出血及支架内血栓发生率。结果在服用同样剂量阿司匹林和氯吡格雷的情况下,3组患者均无大出血及支架内血栓发生,但A组患者皮下出血、牙龈出血发生率明显高于其他两组。结论老年冠心病合并肾功能不全患者介入治疗围术期给予依诺肝素,40mg皮下注射,每早1次,20mg皮下注射,每晚1次,共5～7d,是安全和有效的。     

Deficiency of NG2+ cells contributes to the susceptibility of stroke-prone spontaneously hypertensive rats

Aims: The purpose of this study is to investigate whether the NG2⁺ cells, a class of oligodendrocyte progenitor cells, is involved in the pathophysiology of stroke in stroke‐prone spontaneously hypertensive rat (SHR‐SP). Methods: SHR‐SP, SHR, Wistar‐Kyoto rats (WKY), and C57BJ/6 mice were used. Immunohistochemistry was conducted to evaluate the number of NG2⁺ cells in frozen brain sections. Demyelination was evaluated by Sudan black staining and serum level of myelin basic protein. Middle cerebral artery occlusion (MCAO) was performed to prepare experimental stroke model. Results: The number of NG2⁺ cells was significantly decreased in infarct core and increased in penumbra in WKY rats after MCAO. In brain sections of 6‐month‐old SHR‐SP, the number of NG2⁺ cells was significantly (P < 0.01) less than that in age‐matched SHR and WKY rats. However, this phenomenon was not observed in 3‐month‐old rats. Demyelination was found in 6‐month‐old SHR‐SP but not in 3‐month‐old SHR‐SP. Pharmacological treatment of cuprizone in mice induced demyelination and enlargement of cerebral infarction after MCAO. Conclusion: The decline of NG2⁺ cells may cause demyelination and contribute to the susceptibility of SHR‐SP to ischemic brain injury.     

Intracavernous delivery of a designed angiopoietin-1 variant rescues erectile function by enhancing endothelial regeneration in the streptozotocin-induced diabetic mouse

OBJECTIVE

Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals.

RESEARCH DESIGN AND METHODS

Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days −3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test.

RESULTS

Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of p47^phox and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1–treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1–induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin.

CONCLUSIONS

These findings support the concept of cavernous endothelial regeneration by use of the recombinant Ang1 protein as a curative therapy for diabetic erectile dysfunction.Erectile dysfunction affects up to 75% of all men with diabetes and occurs earlier in such patients than in the general population (1,2). The Massachusetts Male Aging Study revealed that erectile dysfunction is three times as prevalent in diabetic men as in men without diabetes (3). Moreover, men with diabetic erectile dysfunction tend to respond less positively to the currently available oral phosphodiesterase-5 (PDE5) inhibitors than do nondiabetic men (4–6). The reduced responsiveness to PDE5 inhibitors in patients with diabetes may be related to the severity of endothelial dysfunction (7). Because the effects of PDE5 inhibitors rely on endogenous nitric oxide (NO) formation, PDE5 inhibitors may fail to increase the level of cyclic guanosine monophosphate (cGMP) above the necessary threshold if the bioavailable NO is insufficient as a result of severe endothelial dysfunction. Furthermore, PDE5 inhibitors must be used on demand, thus limiting the spontaneity of the sexual act. The use of PDE5 inhibitors is absolutely contraindicated in men who take nitrate compounds because of the risk of extreme hypotension or even death (8). Therefore, new therapeutic strategies are needed.Augmented generation of reactive oxygen species (ROS) is one of the major causes of decreased NO bioavailability in diabetes (9–12). We recently reported in streptozotocin (STZ)-induced type 1 diabetic mice that increased superoxide anion production and peroxynitrite formation in the corpus cavernosum tissue play an important role in the diabetes-induced functional and structural impairments in the cavernous endothelium, such as the decrease in endothelial NO synthase (eNOS) enzyme activity, endothelial cell apoptosis, and subsequent loss of endothelial cell content (12). Therefore, restoring NO bioavailability by inhibiting ROS-mediated endothelial cell damage, regenerating cavernous endothelial cells, or both may serve as promising therapeutic strategies for treating patients with diabetic erectile dysfunction.Various antioxidants have been shown to improve endothelial function and erectile ability in animal models of diabetic erectile dysfunction (10,13,14). Adenovirus-mediated gene transfer of extracellular superoxide dismutase into the corpus cavernosum tissue was shown to reduce superoxide anion levels and raise cavernous cGMP levels, which resulted in partial recovery of erectile function in STZ-induced diabetic rats 2 days after transfection (14). Despite encouraging results in preclinical studies, however, no antioxidants have so far been approved for the treatment of patients with erectile dysfunction. A possible reason is that antioxidant therapy may not be effective if structural damage to cavernous endothelial cells has already progressed. In this regard, regeneration of damaged endothelial cells using angiogenic factors is a more logical approach to treating erectile dysfunction than is the prevention of endothelial cell damage with antioxidants.Local delivery of the vascular endothelial growth factor-A (VEGF-A) gene or protein into the penis has been shown to induce partial or complete recovery of erectile function in STZ-induced type 1 diabetic rats (15–17). However, these studies did not demonstrate whether VEGF-A therapy induces endothelial regeneration. Furthermore, VEGF-A often leads to the formation of disorganized vessels that are leaky, hyperpermeable, and inflamed in experimental systems (18,19), thus greatly compromising the therapeutic utility of VEGF-A. In comparison, angiopoietin-1 (Ang1), the ligand of the Tie2 receptor tyrosine kinase, is a specific growth factor that functions to generate a nonleaky, stable, and functional vasculature (19–22). Transgenic overexpression or gene transfer of Ang1 not only enhances vessel formation but also protects the adult vasculature against vascular leakage (19,23–25). Thus, Ang1 has potential therapeutic applications in angiogenesis and the prevention of vascular leakage. However, our previous study (26) revealed that a single intracavernous delivery of the adenovirus-mediated Ang1 gene failed to induce an angiogenic response in the penis of a hypercholesterolemic rat. Recently, Cho et al. (27) developed a soluble and potent Ang1 variant, cartilage oligomeric matrix protein (COMP)-Ang1, that is more potent than native Ang1 in phosphorylating Tie2 in primary cultured endothelial cells. COMP-Ang1 stimulates angiogenesis with nonleaky neovessel formation in the mouse corneal micropocket assay, whereas VEGF-A stimulates angiogenesis with leaky neovessel formation (27). One mechanism for inducing nonleaky and healthy angiogenesis is the specific activation of Tie2 in endothelial cell-cell or cell-matrix contacts by COMP-Ang1 (28,29).In the current study, we determined the effectiveness of COMP-Ang1 in promoting cavernous endothelial regeneration and restoring erectile function in a mouse model of diabetic erectile dysfunction. In addition, because increased vascular permeability by loss of endothelial cell-cell junction proteins is an important pathophysiological mechanism involved in diabetic retinopathy (30–33), we investigated whether COMP-Ang1 induces nonleaky angiogenesis in the penis by restoring endothelial cell-cell junction proteins.     

Effects of connexin-mimetic peptides on perfusion pressure in response to phenylephrine in isolated,perfused rat kidneys

Background  

Gap junction intercellular communication plays a fundamental role in various tissues and organs. Gap junctions transfer ions and molecules between adjacent cells and are formed by connexins (Cx). It is supposed that vascular conducted responses, which most likely spread through gap junctions in vascular beds, regulate microcirculatory blood flow and maintain vascular resistance. This study provides functional evidence supporting the critical role of gap junctions in a physiological setting and in phenylephrine (PE)-induced vasoconstriction using an ex vivo kidney perfusion technique.