Enhancement of repopulation haemopoiesis by heterozygous connexin 43 stem cells seeded on wild-type connexin 43 stroma

The present paper analyses the results of competitive blood-cell repopulation experiments in which Cx43-WT (connexin 43 wild-type) host mice, whose own HSCs (haemopoietic stem cells) were deleted, were grafted with fetal liver cells: 50% Gpi-1a (glucose phosphate isomerase-1a)/Cx43-WT cells competing with 50% Gpi-1b/Cx43-WT, 50% Gpi-1b/Cx43-HZ (heterozygous) or 50% Gpi-1b/Cx43-KO (knock-out) cells. The percentages of platelets, granulocytes, red cells, B-cells and T-cells containing Gpi-1b in blood samples obtained from 22 to 186 days after grafting, and the percentages of high-proliferation-potential colony-forming cells containing Gpi-1b at 255 days after grafting, were measured. The results show that, if we wait 4 months so that we measure the percentages of Gpi-1b end-cells formed by initially resting stem cells in the graft, values in HZ mice are greater than those in WT and KO mice by 10% or more. We propose a bipolar influence model for blood formation by grafted HSCs to explain this difference and other features of the data. Influence A is a direct one: for individual HSCs, the combined effect on HSC niching and HSC proliferation of Cx43 is superior to that of the KO allele. Influence B is a demographic one: HZ foundation mice compensate by having more HSCs than WT mice. The net outcome of influences A and B is that HZ is the winner.     

心室纤颤时心肌缝隙连接蛋白Cx43的变化

目的 观察心室纤颤(室颤)发生后缝隙连接蛋白Cx43的表达以及缝隙连接改造剂ZP123对Cx43表达的影响.方法 按照随机数字表法将30只家猪分为假手术组、模型组和ZP123干预组,每组10只.以80 V电压持续刺激动物5 s诱发室颤;致颤前15 min ZP123组给予ZP123 1μg/kg静脉推注+ZP12310μg·kg-1·h-1微量泵泵入;模型组泵入生理盐水50 ml;假手术组动物不致颤也不补液.室颤持续8 min后开胸取左心室游离壁心肌,用免疫荧光结合激光共聚焦显微镜技术检测Cx43的分布及水平,用蛋白质免疫印迹法(Western blotting)定量检测Cx43蛋白表达.结果 假手术组Cx43荧光信号强,分布均匀;模型组Cx43荧光信号弱,呈不均一分布;ZP123干预组Cx43荧光信号增强,不均一分布减轻.与假手术组比较,模型组心室肌组织Cx43荧光信号面积百分比、积分吸光度(A)值及蛋白表达均明显下降[面积百分比:(0.64±0.36)%比(1.27±0.19)%,积分A值:15 201±2 613比30 634±4 975,Cx43蛋白表达:0.72±0.08比0.97±0.07,均P＜0.05];与模型组比较,ZP123干预组Cx43表达[面积百分比(0.96±0.16)%,积分A值22 100±4 404,Cx43蛋白表达0.82±0.04]均明显升高(均P＜0.05).结论 室颤发生时心肌组织Cx43表达减少;应用ZP123可减少或逆转Cx43的降解.     

室性心动过速时连接蛋白43含量与分布的变化

目的　探讨室性心动过速 (室速 )时心肌细胞连接蛋白 4 3(Cx 4 3)含量和分布的变化。方法　实验用日本大耳白兔 2 0只 ,随机分为对照组、30min室速组、6 0min室速组、12 0min室速组 4组。分别通过心室刺激复制室性心动过速动物模型 ,应用激光共聚焦显微镜技术和荧光免疫组织化学方法对其发生心律失常心肌连接蛋白 4 3的含量和分布进行定量分析。结果　 30min室速组连接蛋白 4 3像素密度较对照组减少 18.4 % (P <0 .0 5 ) ,6 0min室速组减少 38.0 % (P <0 .0 1) ,12 0min室速组减少 5 4 .8% (P <0 .0 1)。对照组各层间心肌细胞连接蛋白 4 3含量比较无显著差异 ,但心律失常后 ,中间层心肌细胞连接蛋白 4 3含量较其他层心肌细胞含量下降更明显 (P <0 .0 5或P <0 .0 1)。结论　室性心动过速时连接蛋白 4 3迅速降解 ,其分布也发生明显的改变 ,各层心肌细胞连接蛋白 4 3的降解程度也明显不均一。     

AT1 blockade abolishes left ventricular hypertrophy in heterozygous cMyBP‐C null mice: role of FHL1

This research investigated the impact of angiotensin AT1 receptor (Agtr1) blockade on left ventricular (LV) hypertrophy in a mouse model of human hypertrophic cardiomyopathy (HCM), which carries one functional allele of Mybpc3 gene coding cardiac myosin‐binding protein C (cMyBP‐C). Five‐month‐old heterozygous cMyBP‐C knockout (Het‐KO) and wild‐type mice were treated with irbesartan (50 mg/kg/day) or vehicle for 8 weeks. Arterial blood pressure was measured by tail cuff plethysmography. LV dimension and function were accessed by echocardiography. Myocardial gene expression was evaluated using RT‐qPCR. Compared with wild‐type littermates, Het‐KO mice had greater LV/body weight ratio (4.0 ± 0.1 vs. 3.3 ± 0.1 mg/g, P < 0.001), thicker interventricular septal wall (0.70 ± 0.02 vs. 0.65 ± 0.01 mm, P < 0.02), lower Mybpc3 mRNA level (?43%, P < 0.02), higher four‐and‐a‐half LIM domains 1 (Fhl1, +110%, P < 0.01), and angiotensin‐converting enzyme 1 (Ace1, +67%, P < 0.05), but unchanged Agtr1 mRNA levels in the septum. Treatment with irbesartan had no effect in wild‐type mice but abolished septum‐predominant LV hypertrophy and Fhl1 upregulation without changes in Ace1 but with an increased Agtr1 (+42%) in Het‐KO mice. Thus, septum‐predominant LV hypertrophy in Het‐KO mice is combined with higher Fhl1 expression, which can be abolished by AT1 receptor blockade, indicating a role of the renin‐angiotensin system and Fhl1 in cMyBP‐C‐related HCM.     

自体骨髓间充质干细胞移植后心肌缝隙连接蛋白43表达与室性心动过速发生的关系

目的:缝隙连接蛋白43对维持心肌细胞的连接通讯功能、电信号传导和正常的节律性收缩起重要作用,其表达和分布的异常是多种室性心律失常的解剖学基础,建立小型猪急性心肌梗死模型.观察自体骨髓间充质干细胞移植后室性心动过速的发生及心肌缝隙连接蛋白43的表达方法:实验于2006-01/2007-01在河北省人民医院导管室完成。①材料:选取8～12月龄小型猪22头,由河北医科大学实验动物中心提供,体质量20～30 kg,随机数字表法分为细胞移植组12头、模型对照组10头.实验过程中对动物的处置符合动物伦理学际准②实验方法:无菌条件下抽取猪双侧股骨骨髓20 mL,percoll法 贴壁法分离培养骨髓间充质干细胞,待细胞生长达75%融合时用胰酶消化传代。将传至第2代细胞加入终浓度为10μmol/L的5-氮胞苷进行诱导,用胶体金标记12 h后继续培养20 d用于移植:两组小型猪均采用球囊堵闭法建立急性心肌梗死模型.心电图监测示相关至少2个导联ST段抬高大于0.2 mV、术后血肌钙蛋白和肌酸磷酸激酶同工酶升高超过正常的两倍为建模成功标准:细胞移植组于造模成功后经OTW球囊于第一对角支远端1 cm处再次阻断血流,注入经胶体金标记的10×10~7个骨髓间充质干细胞。③实验评估:于细胞移植后2 h及4周行电生理程序刺激.观察室性心动过速的发生情况。末次电生理检查后、采用免疫组化染色法检测心肌缝隙连接蛋白43的表达.计算其积分吸光度值。结果:①模型建立指标检测:与术前比较.造模后所有小型猪血肌钙蛋白含量和肌酸磷酸激酶同工酶活性均增高,峰值浓度分别为(21.3±3.6)μg/L和(178.3×41.4)IU/L,术中心电图ST段平均抬高(10.67±1.43)mm.证明急性心肌梗死模型成功建立。②骨髓间充质干细胞移植后室性心动过速的发生情况:与模型对照组诱发出室性心动过速的动物数量比较,术后2 h细胞移植组无明显变化(X~2=0.201,P=0.650),术后4周细胞移植组明显降低(X~2=4.455.P=0.035)。②骨髓间充质干细胞移植后梗死心肌缝隙连接蛋白43的表达:术后4周移植到梗死心肌的骨髓间充质干细胞与宿主心肌生长为一体,移植部位颜色变黑,苏木精-伊红染色示移植细胞的胞浆呈紫红色。细胞移植组心肌梗死区缝隙连接蛋白43积分吸光度值明显高于模型对照组(t=16,82.P=0.00),细胞移植组中未发生室性心动过速小型猪的梗死心肌缝隙连接蛋白43的表达明显高于发生室性心动过速小型猪(t=5.06,P=0.00)。结论:自体骨髓间充质干细胞移植可促进急性心肌梗死猪心肌缝隙连接蛋白43的表达,其表达程度可能与急性心肌梗死室性心动过速的发生有关。     

缝隙连接蛋白Cx32与Cx43在癫痫发病中的作用

目的:探讨缝隙连接蛋白(Cx)与癫痫之间的关系.方法:采用免疫组织化学方法测定氯化锂-匹罗卡品致痫大鼠癫痫发作后不同时间不同脑区Cx32与Cx43免疫阳性表达情况.结果:与对照组比较,致痫组大鼠海马区与皮层区Cx32和Cx43免疫阳性表达在癫痫发作1h后开始增强(Cx32免疫阳性细胞数分别为海马区16.62±4.51和皮层区14.85±3.30,均P＜0.05;Cx43免疫阳性细胞数分别为海马区18.26±4.03和皮层区18.65±4.51,均P＜0.01),24 h达到高峰(Cx32免疫阳性细胞数分别为海马区46.53±9.47和皮层区28.25±8.69,均P＜0.01;Cx43免疫阳性细胞数分别为海马区39.77±7.79和皮层区26.50±6.56,均P＜0.01),此后逐步下降.但至14 d Cx32海马区与皮层区免疫阳性细胞数分别为22.45±6.56和15.92±3.16,仍高于对照组(均P＜0.05),而Cx43在14 d的表达则有所不同:海马区免疫阳性细胞数17.54±3.77仍高于对照组(P＜0.01);皮层区表达则降至正常水平.致痫24 h时海马区Cx32和Cx43免疫阳性表达均明显高于同一时限的皮层区(均P＜0.05).结论:Cx32和Cx43参与了癫痫的发生与发展过程,反映了脑组织中神经元、星形胶质细胞之间的缝隙连接与癫痫发病机制密切相关.     

Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice

Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1(-/-)) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1(-/-) mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1(-/-) mice. Similarly, the right ventricles were more dilated in HO-1(-/-) mice. After seven weeks of hypoxia, only HO-1(-/-) mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1(-/-), but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension. J.Clin. Invest. 103:R23-R29 (1999).     

Improved insulin-sensitivity in mice heterozygous for PPAR-gamma deficiency

The thiazolidinedione class of insulin-sensitizing, antidiabetic drugs interacts with peroxisome proliferator-activated receptor gamma (PPAR-gamma). To gain insight into the role of this nuclear receptor in insulin resistance and diabetes, we conducted metabolic studies in the PPAR-gamma gene knockout mouse model. Because homozygous PPAR-gamma-null mice die in development, we studied glucose metabolism in mice heterozygous for the mutation (PPAR-gamma(+/-) mice). We identified no statistically significant differences in body weight, basal glucose, insulin, or FFA levels between the wild-type (WT) and PPAR-gamma(+/-) groups. Nor was there a difference in glucose excursion between the groups of mice during oral glucose tolerance test, but insulin concentrations of the WT group were greater than those of the PPAR-gamma(+/-) group, and insulin-induced increase in glucose disposal rate was significantly increased in PPAR-gamma(+/-) mice. Likewise, the insulin-induced suppression of hepatic glucose production was significantly greater in the PPAR-gamma(+/-) mice than in the WT mice. Taken together, these results indicate that - counterintuitively - although pharmacological activation of PPAR-gamma improves insulin sensitivity, a similar effect is obtained by genetically reducing the expression levels of the receptor.     

Phenotypic expression of heterozygous lipoprotein lipase deficiency in the extended pedigree of a proband homozygous for a missense mutation.

Familial lipoprotein lipase (LPL) deficiency is a rare genetic disorder accompanied by well-characterized manifestations. The phenotypic expression of heterozygous LPL deficiency has not been so clearly defined. We studied the pedigree of a proband known to be homozygous for a mutation resulting in nonfunctional LPL. Hybridization of DNA from 126 members with allele-specific probes detected 29 carriers of the mutant allele. Adipose tissue LPL activity, measured previously, was reduced by 50% in carriers, but did not reliably distinguish them from noncarriers. Carriers were prone to the expression of a form of familial hypertriglyceridemia characterized by increased plasma triglyceride, VLDL cholesterol and apolipoprotein B, and decreased LDL and HDL cholesterol concentrations. These manifestations were age modulated, with conspicuous differences between carriers and noncarriers observed only after age 40. Several noncarriers exhibited similar lipid abnormalities, but without the inverse relationship between VLDL cholesterol and LDL cholesterol noted among carriers. In addition to age and carrier status, the potentially reversible conditions, obesity, hyperinsulinemia and lipid-raising drug use were contributory. Thus heterozygous lipoprotein lipase deficiency, together with age-related influences, may account for a form of familial hypertriglyceridemia.     

Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption.

With the aim of establishing whether a genetically reduced capability of producing apolipoprotein E (apo E) can affect atherogenesis, we have compared the consequences of dietary stress on normal mice and on mice heterozygous or homozygous for a disrupted apo E gene. A dramatically accelerated development of lesions occurred in the vasculature of the homozygous mutants as a result of feeding an atherogenic diet for 12 wk, and extensive deposition of lipid-filled macrophages was found outside the cardiovascular system. In nine heterozygotes fed the atherogenic diet for 12 wk, the amount of apo E in their total plasma lipoproteins increased to a level comparable to normal, but all nine developed much larger foam cell lesions in their proximal aorta than those found in 3 of 9 normal mice fed the same diet. The other six normals had no lesions. Our study demonstrates that heterozygous mice with only one functional apo E gene are more susceptible to diet-induced atherosclerosis than are normal, two-copy mice. Genetically determined quantitative limitations of apo E could, therefore, have similar effects in humans when they are stressed by an atherogenic diet.     

急性心房颤动犬缝隙连接蛋白40和43重构及胺碘酮的干预作用

目的 采用快速心房起搏致犬急性心房颤动(简称房颤)模型,观察心房肌缝隙连接蛋白40和43(Cx40、Cx43)含量的改变以及胺碘酮干预的效果.方法 18只成年杂种犬,随机等分为三组,即正常对照组、急性房颤组、胺碘酮组.快速心房起搏,对照组不起搏,胺碘酮组先静脉注射胺碘酮负荷量3 mg/kg,10～15 min内注入,后以维持量1.5 mg/kg静脉滴注进行干预,另两组给予等容量的生理盐水.连续刺激并且房颤8 h后,取右心耳组织,用Western blot 检测Cx40和Cx43的含量,用Lucifer Yellow划痕标记荧光传输技术在荧光显微镜上观察细胞缝隙连接通讯状态.结果 急性房颤组心房肌组织Cx40和Cx43含量较对照组降低(P均<0.05),胺碘酮组Cx40、Cx43含量较正常对照组低,差异有显著性(P均<0.05),较房颤组含量差异无显著性(P>0.05),胺碘酮可以改善急性房颤时细胞缝隙连接通讯的传导.结论 急性房颤犬缝隙连接蛋白40和43发生了结构重构,从而影响细胞间通讯,胺碘酮可以改善细胞间电信号传导,但不能改善缝隙连接结构重构.     

A compound heterozygous mutation in glycoprotein VI in a patient with a bleeding disorder

Summary.  Background: The physiological relevance of the collagen glycoprotein VI (GPVI) receptor was known prior to its recognition as a platelet membrane receptor as several patients lacking GPVI as a consequence of autoantibody inhibition presented with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation have not yet been identified. Results: In the present study, we describe a patient with a lifelong history of bleeding problems, structurally normal platelets but a functional platelet defect. Platelet aggregations are normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide (CRP). ATP dense granule secretion is normal with ADP but absent with Horm collagen. Thrombus formation on a collagen surface in flowing blood is reduced but more single platelets are attached. Remarkably, the platelet function analyzer-100 shows a shortened collagen/ADP closure time. Flow cytometry demonstrates an absent expression of GPVI whereas immunoblot analysis shows strongly reduced levels of GPVI. The patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. The parents without clinical bleeding problems are heterozygous carriers. The mother carries the S175N mutation and presents with a mild functional platelet defect. In vitro studies show a reduced membrane expression and convulxin binding with the mutated S175N compared with the wild-type (WT) GPVI receptor. Conclusions: This study presents the first patient with a proven genetic GPVI defect.     

Diastolic ventricular dysfunction as a marker for hypertrophic cardiomyopathy in a family with a novel alpha-tropomyosin mutation.

BACKGROUND: Early identification of familial cases of hypertrophic cardiomyopathy (HCM) depends on screening echocardiography, but hypertrophy may not be the most sensitive marker for the disease. We report the echocardiographic findings of a family with HCM and a newly reported mutation in the gene (TPM1) encoding alpha-tropomyosin.Methods and results An 8-year-old girl had sudden cardiac death, and was found to have HCM and a novel L185R-TPM1 mutation on postmortem examination. Screening echocardiograms and DNA analyses were performed on her family. Of the 5 remaining family members, 3 were genetically affected. Those without the TPM1 mutation had normal echocardiographic results. The only echocardiographic finding that identified all 3 of the gene-positive family members was an abnormal left ventricular diastolic filling pattern. CONCLUSION: Abnormal left ventricular diastolic filling patterns, indicating diastolic dysfunction, may provide an early marker for the diagnosis of familial HCM in children, even in the absence of left ventricular hypertrophy.     

抗凝血酶基因杂合突变导致的抗凝血酶缺陷症

目的 对1例先天性抗凝血酶（AT）缺陷症患者及其家系成员进行AT表型及基因突变检测。方法 采用发色底物法和免疫比浊法分别检测先证者及其家系成员血浆AT活性（AT：A）和AT抗原含量（AT：Ag），并采用PCR法对先证者AT基因的7个外显子及其侧翼内含子序列进行扩增，PCR产物纯化后直接测序检测基因突变。结果 先证者的AT：Ag正常，但AT：A为正常值的65％，表现为Ⅱ型AT缺陷，其AT基因外显子6区第13830位核苷酸发生了G—A杂合突变，引起Arg393His错义突变。同样突变也见于该家系其他3名成员。结论 该家系成员的Ⅱ型AT缺陷由AT基因G13830A杂合突变所致，可致血栓形成。     

The heme oxygenase inducer hemin protects against cardiac dysfunction and ventricular fibrillation in ischaemic/reperfused rat hearts: role of connexin 43

Cardiac expression of cytoprotective gene heme oxygenase‐1 (HO‐1) is modulated by ischaemia and reperfusion (I/R). We therefore hypothesized that pretreatment with hemin, an inductor of HO‐1, would precondition the heart against post‐ischaemic dysfunction and ventricular fibrillation (VF). Male Wistar rats were given either hemin or HO enzyme inhibitor zinc protoporphyrin IX (ZnPP IX). Isolated hearts were subjected to 30?min global ischaemia followed by 120?min of reperfusion or were aerobically perfused in a time‐matched non‐ischaemic protocol. Control animals received no pretreatment. Compared to non‐perfused controls, pretreatment with hemin increased HO‐1 mRNA 13‐fold (p<0.001) and HO‐1 protein 3.5‐fold (p?0.001), improved post‐ischaemic aortic flow, coronary flow, LVDP and ?Dp/dt (p<0.01) and decreased LVEDP (p<0.001) and the incidence of VF (p = 0.001). The improved post‐ischaemic cardiac function and reduction of VF were accompanied by a higher total connexin 43 (Cx43) level compared to non‐pretreated and ZnPP IX pretreated hearts, and accumulation of non‐phosphorylated gap junction protein Cx43 in intercalated discs and lateral plasma membrane of cardiomyocytes. Cardioprotection by HO‐1 appeared to be independent of cGMP. Administration of ZnPP IX had no effect on cardiac function or VF. Our results show that pharmacological modulation of HO‐1 pathway may provide a new therapeutic approach to protect the heart against post‐ischaemic dysfunction and I/R‐induced VF possibly by a Cx43 dependent mechanism.     

The heme oxygenase inducer hemin protects against cardiac dysfunction and ventricular fibrillation in ischaemic/reperfused rat hearts: role of connexin 43

Cardiac expression of cytoprotective gene heme oxygenase-1 (HO-1) is modulated by ischaemia and reperfusion (I/R). We therefore hypothesized that pretreatment with hemin, an inductor of HO-1, would precondition the heart against post-ischaemic dysfunction and ventricular fibrillation (VF). Male Wistar rats were given either hemin or HO enzyme inhibitor zinc protoporphyrin IX (ZnPP IX). Isolated hearts were subjected to 30 min global ischaemia followed by 120 min of reperfusion or were aerobically perfused in a time-matched non-ischaemic protocol. Control animals received no pretreatment. Compared to non-perfused controls, pretreatment with hemin increased HO-1 mRNA 13-fold (p<0.001) and HO-1 protein 3.5-fold (p相似文献