体外扩增的骨髓基质细胞在缺血心肌内的存活及对心肌重塑影响

目的观察体外扩增骨髓基质细胞(MSCs)在缺血心肌内存活以及对缺血心肌重塑的影响.方法兔下肢骨获取MSCs,体外扩增传代,收获第二代细胞行,4,6二氨基-2-苯茚二酮(DAPI)标记.结扎前降支制作心肌梗死动物模型,3d后2×106DAPI标记MSCs直接注射到实验组缺血坏死区心肌(n=8),对照组心肌注射相同体积培养基(n=6).细胞植入前、植入后1周、4周心脏超声检查,4周时收获心肌标本,计算缺血范围与梗死面积.荧光显微镜观察植入的DAPI标记MSCs在缺血区存活情况,Masson's染色对比实验组与对照组缺血坏死区瘢痕化程度.结果荧光显微镜检发现植入的MSCs在缺血区存活增殖并融合于缺血区心肌;心脏超声显示细胞移植组心室容积变化显著小于对照组(△EDV 0.27±0.11ml vs 0.46±0.14ml P＜0.05;△EDV 0.17±0.13mlvs 0.32±0.11 ml P＜0.05);细胞移植组局部室壁运动情况明显优于对照组;4周后,细胞移植组平均缺血范围与梗死区面积与对照组比较明显减小(14.70±0.04%vs19.6±0.09%P＜0.05;4.43±1.0%vs13.72±0.92%P＜0.05);细胞移植组缺血坏死区纤维化程度较轻.结论体外扩增的MSCs可在缺血坏死区存活并融合于植入区心肌,植入细胞显著减轻缺血坏死区心肌重塑.     

小鼠骨髓基质细胞参与缺血组织血管再生及时相关系

目的探讨体外培养的骨髓基质细胞体内移植后在缺血区新血管生成中的作用及其时相关系.方法分离5～6周龄的小鼠胫骨、股骨,用预冷的DMEM/F12培养基冲洗出骨髓,经密度梯度离心分离出骨髓单个核细胞体外培养.体外成内皮细胞诱导分化鉴定分离的细胞,建立下肢缺血模型,荧光标记的体外扩增的骨髓基质细胞移植入左侧缺血组织,右侧注射等量培养介质作为对照组,分为7d、14d、28d 3个观察期(n=6),冰冻切片荧光显微镜观察,毗邻切片HE及vWF染色,检查荧光阳性细胞与染色阳性细胞的时空关系,并计算移植前后毛细血管密度的变化.结果体外分离培养的骨髓基质细胞在成内皮细胞供及物存在条件下表达vWF,并分泌NO.移植后7d,观察到大量分散开来的DAPI标记细胞存活(蓝色荧光),毗邻切片HE染色显示了高密度HE染色阳性区和荧光阳性区的一致性,MSC来源的供体细胞核浆比大,提示是较幼稚细胞,移植区未见明显炎症反应.移植后14d,组织切片可见大量荧光标记细胞存活,呈与骨骼肌直径大小相似的网状散在分布,对照组未见荧光,在毗邻的切片,大部分荧光阳性细胞vwF因子染色阳性,毗邻切片的HE染色,细胞移植组骨骼肌纤维未见明显异常,而对照组骨骼肌可见部分肌纤维变性.移植的28d,移植组毛细胞血管数量为(57±18)mm2,对照组为(36±12)mm2,移植组高于对照组(P＜0.05).结论体外培养的骨髓基质细胞在体外能够定向分化为血管内皮细胞,移植入体内缺血区能促进缺血区新血管生成,可望用作组织缺血细胞治疗的种子细胞.     

体外扩增骨髓基质细胞在缺血心肌内的存活及对心肌重塑影响

目的:观察体外扩增骨髓基质细胞(MSCs)在缺血心肌内存活以及对缺血心肌重塑的影响。方法:兔下肢骨获取MSCs,体外扩增传代,收获第2代细胞行4,6二氨基2苯茚二酮(DAPI)标记。结扎前降支制作心肌梗死动物模型,3d后将2×106DAPI标记MSCs直接注射到实验组(8只)缺血坏死区心肌,对照组(6只)心肌注射相同体积培养基。细胞植入前,植入后1、4周心脏超声检查,4周时收获心脏标本,计算缺血范围与梗死面积。荧光显微镜观察植入的DAPI标记MSCs在缺血区存活情况,Masson染色对比实验组与对照组缺血坏死区瘢痕化程度。结果:荧光显微镜检发现植入的MSCs在缺血区存活增殖并融合于缺血区心肌;心脏超声显示实验组心室容积变化显著小于对照组[ΔEDV(0.27±0.11)∶(0.46±0.14)ml,P<0.05;ΔESV(0.17±0.13)∶(0.32±0.11)ml,P<0.05];实验组局部室壁运动情况明显优于对照组;4周后,实验组平均缺血范围与梗死区面积与对照组比较明显减小[(14.70±0.04)∶(19.6±0.09)%,P<0.05;(4.43±1.0)∶(13.72±0.92)%,P<0.05];实验组缺血坏死区纤维化程度较轻。结论:体外扩增的MSCs可在缺血坏死区存活并融合于植入区心肌,植入细胞显著减轻缺血坏死区心肌重塑。     

骨髓基质细胞移植治疗扩张型心肌病可行性的探讨

目的 :有研究表明骨髓基质细胞移植入心肌后可向心肌细胞方向转化。我们探讨骨髓基质细胞移植对阿霉素诱导的扩张型心肌病模型的作用。方法 :新西兰大白兔 2 0只 ,实验组每周给阿霉素注射 2mg kg ,共 8周 ,建立扩张型心肌病模型。第 8周时取兔自身骨髓基质细胞培养 ,第 10周培养细胞行DAPI(6 diamidino 2 phenylindone)标记 ,并植入自身心肌组织 ,超声心动图监测心功能状况。结果 :与病理模型组相比 ,骨髓基质细胞移植后 2周可见移植局部心肌组织中有移植的细胞存活 ,组织学检查结果显示移植局部心肌损伤情况要轻于病理模型组。细胞凋亡指数也明显少于病理模型组。超声心动图示移植局部心肌运动速度由 (2 1± 0 5 1)cm s提高至 (3 8± 0 4 7)cm s(P <0 0 5 ) ,但整体心功能未见改善。结论 :自体骨髓基质细胞移植对扩张型心肌病的治疗有良好的前景 ,但在细胞的体外处理、移植细胞的方式、数量、及其作用机理方面都需要进一步的探讨     

经静脉移植自体骨髓单个核细胞治疗猪急性心肌梗死的实验研究

目的探讨静脉内移植自体骨髓单个核细胞(BM-MNCs)治疗猪急性心肌梗死的有效性和安全性。方法静脉内移植BM-MNCs后4周观察移植细胞归巢情况、小血管密度、心功能及冠脉侧支血管形成情况及可能发生的不良反应。结果静脉内MNCs移植后4周心肌细胞间可见少量发蓝色荧光的移植细胞,散在分布,心肌细胞间有较多新生毛细血管并可见新生的心肌样细胞;移植组小血管密度(68.25±9.54)条/mm~2明显高于对照组(35.14±7.06)条/mm~2(P<0.01),左心室射血分数(LVEF,62.75%±5.08%)、短轴缩短率(FS,40.28%±4.73%)也明显高于对照组LVEF(46.34%±5.94%)、FS(29.34%±3.86%)(均为P<0.05);移植组冠脉侧支循环形成较对照组明显;静脉内移植MNCs没有发现异常增生、钙化或肿瘤形成。结论经静脉移植的BM-MNCs可归巢到宿主心肌,有助于促进缺血心肌血管新生,改善左心室收缩功能的作用。     

骨髓细胞移植对心肌梗死后大鼠心肌细胞凋亡及Bcl-2、Bax表达的调节

目的　观察骨髓单个核细胞 (BM MNCs)移植对心肌梗死 (MI)后恢复期心肌细胞凋亡及心功能的影响 ,并探讨其可能机制。方法　通过结扎左冠状动脉前降支制成MI模型。 2周后第二次开胸在心外膜下梗死区及周边区多点注射BM MNCs混悬液 2 0 0 μl(5× 10 6 个细胞 )。经超声心动图测定左室射血分数 (EF)、短轴缩短率 (FS)等指标。采用DNA缺口末端标记法 (TUNEL)检测心肌细胞凋亡 ,免疫组化法检测Bcl 2、Bax蛋白在心肌细胞中表达水平。结果　术后 4周 ,移植组EF、FS值均高于对照组 (P <0 0 5 ) ;8周时EF、FS值升高达 2 2 4 %、2 8 1% (P <0 0 5 )。TUNEL结果显示心肌细胞凋亡指数在移植组明显低于对照组 (4周 :0 0 94 6± 0 0 17vs 0 173± 0 0 18,P <0 0 5 ;8周 :0 0 916± 0 0 14vs 0 182± 0 0 15 ,P <0 0 5 )。免疫组化结果表明 ,移植组Bcl 2蛋白表达高于对照组 (4周 :12 6 6± 3 37vs 5 6 8± 2 5 3,P <0 0 5 ;8周 :13 0 8± 3 0 87vs 5 0 2± 1 91,P <0 0 5 ) ;Bax蛋白表达则降低 (4周 :11 4 8± 3 134vs 2 0 12± 3 91,P <0 0 5 ;8周 :9 98± 3 0 3vs 2 2 74± 3 12 ,P <0 0 5 )。结论　骨髓单个核细胞移植可抑制MI后心肌细胞凋亡的发生 ,从而保护心功能 ,其机制可能与对Bcl 2、     

心肌内移植自体骨髓单个核细胞治疗急性心肌梗死的实验研究

目的探讨心肌内移植自体骨髓单个核细胞(BM-MNCs)治疗猪急性心肌梗死的有效性和安全性。方法11只小型猪分为两组:BM-MNCs组(n=6)和对照组(n=5)。心肌内移植BM-MNCs后4周观察移植细胞归巢情况、小血管密度、心功能及冠脉侧支血管形成情况及可能发生的不良反应。以超声心动图评价心室结构及功能变化。结果心肌内MNCs移植后4周心肌细胞间可见发蓝色荧光的移植细胞,心肌细胞间有较多新生毛细血管并可见新生的心肌样细胞。移植组小血管密度、左室射血分数(LVEF)及短轴缩短率(FS)明显高于对照组(P<0.01)而左室舒张末期内径(LVEDd)低于对照组(P<0.05)。移植组冠脉侧支循环形成较对照组明显。心肌内移植MNCs没有发现异常增生、钙化或肿瘤形成。结论心肌内移植BM-MNCs有较多的移植细胞归巢到宿主心肌,有助于促进缺血心肌血管新生,有改善左室收缩及舒张功能,减轻心室重构的作用。     

骨髓动员在糖尿病小鼠缺血肢体新生血管形成中的作用

目的探讨骨髓动员在糖尿病小鼠缺血肢体新生血管形成中的作用。方法采用链脲佐菌素建立糖尿病鼠模型,切除一侧肢体股动脉,注射粒细胞集落刺激因子(G-CSF)进行骨髓动员,以观察缺血肢体新生血管的形成。结果糖尿病小鼠模型建立8周后,血浆一氧化氮(NO)水平开始下降,骨髓动员后8 d,外周血CD~+_(34)细胞明显增加[从(0．15±0．05)%到(2．30±0．41)%],半定量RT-PCR检测缺血肢体肌肉组织的血管内皮细胞生长因子mRNA表达与正常对照组比较明显增强；动员后2周,激光多普勒血流检测骨髓动员组的缺血肢体血流明显高于未动员组[(76．37±6．10)%vs(18．07±3．40)%,P＜0．05],vWF免疫组化微血管密度检测动员组也明显高于未动员组[(26．6±4．8)个/每视野vs(11．5±2．6)个/每视野,P＜0．05]。结论糖尿病能降低小鼠血管内皮细胞的功能,但骨髓动员能促进糖尿病小鼠缺血肢体的新生血管形成。     

倒卵叶五加总皂甙对大鼠心肌缺血/再灌注损伤后细胞凋亡的作用及机制

目的　探讨倒卵叶五加总皂甙 (SAOH)对大鼠缺血心肌再灌注后细胞凋亡的作用。方法　采用结扎左冠状动脉前降支再灌注复制大鼠心肌缺血再灌注损伤 (IRI)的模型 ,结扎前 10 min舌下静脉注射 SAOH,测定心肌缺血3 0 min后再灌注 6h心肌组织丙二醛 (MDA)和血浆超氧化歧化酶 (SOD)的含量 ,并用末端脱氧核苷酸转移酶介导的带生物素的 d UTP缺口末端标记 (TUNEL)的方法及免疫组化观察左心室心肌细胞凋亡及凋亡相关基因 Bcl- 2和 Bax的蛋白表达。结果　再灌注 6h后 ,SAOH1 组 (5 0 mg/ kg)及 SAOH2 组 (10 0 mg/ kg)较 IRI组 ,心肌组织 MDA含量明显减少 [(4.89± 0 .2 6)、(3 .5 7± 0 .2 1) nm ol/ mg· pro vs(6.97± 0 .3 4) nmol/ mg· pro,P值均 <0 .0 1];血浆 SOD活性明显提高 [(9.88± 0 .18)、(11.40± 0 .3 7)μIU / L vs (3 .3 9± 0 .2 4)μIU / L ,P值均 <0 .0 1];心肌细胞凋亡指数 (AI)显著减少 [(14.67± 3 .0 5 ) %、(7.49± 2 .62 ) % vs(2 1.96± 7.68) % ,P值均 <0 .0 1];Bax蛋白表达明显减少 [(7.73± 1.84) %、(5 .0 2± 1.0 1) % vs (18.5 9± 5 .68) % ,P值均 <0 .0 1];而心肌 Bcl- 2蛋白的表达显著增加 [(12 .5 1± 3 .42 ) %、(17.48± 4.72 ) % vs(7.3 2± 2 .0 5 ) % ,P值 <0 .0 5 ,<0     

经冠状动脉移植自体BM-MNCs治疗猪急性心肌梗死的作用

目的探讨冠状动脉内移植自体骨髓单个核细胞（BM—MNCs）治疗猪急性心肌梗死的有效性。方法实验动物分为冠脉内移植MNCs组（n=6）及冠脉对照组（n=5）。冠脉内移植BM—MNCs后4周观察移植细胞归巢情况、小血管密度、心功能变化。结果冠脉内MNCs移植后4周,心肌细胞间可见发蓝色荧光的移植细胞,散在分布,心肌细胞间有较多新生毛细血管并可见新生的心肌样细胞。移植组小血管密度、左室射血分数（LVEF）及短轴缩短率（FS）明显高于对照组（P〈0．01）,左室舒张末期内径（LVEDd）低于对照组（P〈0．05）。结论经冠脉移植的BM—MNCs可归巢到宿主心肌,有促进缺血心肌血管新生、改善左室收缩功能、减轻心室重构的作用。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.