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.     

骨髓基质细胞的血管新生作用及对心功能的影响

目的　观察骨髓基质细胞在缺血心肌内诱导血管新生的作用及对心功能的影响。方法　复制兔心肌梗死动物模型 ,3d后将体外分离扩增的自体骨髓基质细胞用荧光标记物DAPI标记后移植到梗死周围缺血区心肌 (细胞移植组 ,n =8) ,对照组注射等量培养基 (n =6 )。 4W后 ,先进行心脏超声检查分别测定对照组和细胞移植组左室射血分数 (EF)和短轴缩短率 (FS) ,然后采集移植区心肌标本 ,用荧光示踪方法观察移植细胞在缺血坏死区心肌存活的情况 ,用CD31单克隆抗体免疫组化染色法测定毛细血管密度。结果　细胞移植组心肌组织和小血管内壁均可见蓝色荧光的DAPI标记的移植细胞 ;细胞移植组心肌毛细血管密度显著高于对照组 [(16 3 0 0± 2 5 85 )vs (96 0 0± 16 6 1) ,P <0 0 5 ];细胞移植组EF较对照组增高 [(0 5 0± 0 0 3)vs(0 4 6± 0 0 3) ,P <0 0 5 ],短轴缩短率FS也高于对照组[(2 7 0 2± 1 2 7)vs (2 3 85± 1 6 9) ,P <0 0 5 ]。结论　体外扩增的骨髓基质细胞可在缺心肌内存活 ,并能促进缺血组织血管新生、改善心功能。     

骨髓间充质干细胞移植对心肌梗死大鼠左心功能的影响

目的研究不同途径和不同浓度的骨髓间充质干细胞(MSCs)移植对心肌梗死大鼠左心功能的影响。方法结扎左冠状动脉前降支制备大鼠心肌梗死模型,采用同种异体大鼠MSCs体外分离、纯化、扩增,4’,6-二脒-2-苯基吲哚(DAPI)标记,将48只SD大鼠随机分为6组,A、B、C和D组于心肌梗死后2周经尾静脉移植MSCs(A组:细胞数5×105、B组:细胞数1×106、C组:细胞数5×106、D组:细胞数1×107),E组:经心外膜注射MSCs(细胞数5×106),对照组:注入等量培养基。4周后测定左心功能并行免疫组织化学检测。结果(1)经静脉和心外膜注射移植MSCs大鼠心肌组织中均可以观察到DAPI标记细胞存在,免疫组织化学阳性标记细胞α-肌动蛋白检测阳性,与对照组比较,两组左心功能均明显改善(P<0.05)和梗死面积均明显缩小[分别为(45±2)%,(40±4)%和(41±3)%,P<0.05]。(2)与对照组比较,A组左心功能无明显改善(P>0.05),B、C、D组左心功能明显改善(P<0.05),C和D组优于B组(P<0.05)。结论MSCs经不同途径归巢至心肌梗死区,改善心功能。     

骨髓基质细胞在缺血心肌内存活及血管新生作用

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其诱导血管新生作用. 方法无菌条件下从兔股骨和胫骨抽取抗凝骨髓,采用密度梯度离心获取单个核细胞,接种于DMEM/F-12培养基差速贴壁粘附纯化MSCs,建立体外培养体系,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.     

骨骼肌细胞移植对缺血坏死心肌的修复作用

目的观察兔自体骨骼肌细胞移植给坏死心肌后的分化状态及其对坏死心肌的修复作用和对心功能的改善情况。方法将20只成年兔分为移植组和对照组,取臀肌进行骨骼肌细胞的体外分离和培养,结扎兔的左冠状动脉制成缺血坏死心肌模型。1周后,给兔坏死的心肌部位注射骨骼肌细胞悬液,对照组注射等量的培养液。移植1个月后,通过心脏超声对兔心功能进行检查并取材,对2组坏死心肌进行HE染色,光镜观察,免疫组化分析。结果移植后4周心脏超声检查结果,移植组与对照组左心室射血分数分别为(65.00±1.25)%和(55.00±2.35)%,左室后壁收缩期末厚度分别为(3.21±0.34)和(2.13±0.36)mm,左室后壁舒张期末厚度分别为(2.20±0.45)和(1.54±0.24)mm,2组比较差异均有统计学意义(t值分别为10.080、1.915、3.603,P<0.05)。移植组坏死的心肌及其边缘可见多核的肌细胞生长,myosin免疫组化提示有骨骼肌细胞;对照组可见心肌细胞排列紊乱,纤维化明显,无多核细胞形成。结论骨骼肌细胞移植可以对坏死心肌起修复作用,并能改善心功能状态。     

骨髓间充质干细胞不同移植方法对心肌梗死大鼠心脏的保护作用

目的对比研究经尾静脉和心外膜直视注射移植骨髓间充质干细胞（MSCs）对心肌梗死大鼠的心脏保护作用。方法结扎左冠状动脉前降支制备大鼠心肌梗死模型,采用同种异体MSCs体外分离、纯化、培养和标记,在梗死后2周,A组经尾静脉植入MSCs,B组心外膜直视注射MSCs,C组尾静脉注入等量DMEM培养液为对照。4周后测定心功能,并行免疫组织化学检测。结果移植组心肌组织中可以观察到DAPI标记细胞存在,免疫组化显示阳性标记细胞α-Actin肌动蛋白检测阳性。与对照組相比,A和B组左心功能显著改善（P<0.05）,A和B组梗死面积明显缩小[分别为（45±2）%,（40±4）%和（41±3）%,P<0.05]。结论MSCs经静脉移植和心外膜植入都能够归巢至心肌梗死区,对梗死大鼠的心脏都具有保护作用。     

经静脉和经梗死心肌周边移植自体骨髓间质干细胞对心肌梗死后心功能影响的比较研究

目的比较经静脉移植和心肌移植骨髓间质干细胞(MSCs)对心肌梗死后心功能的影响。方法结扎30只日本大耳兔的冠状动脉左前降支,建立急性心肌梗死模型后分成3组:损伤对照组、静脉移植组和心肌移植组。5周后检测各移植组抗DAPI染色及心脏的结构和功能。结果静脉移植组、心肌移植组在移植区均可检测到大量DAPI阳性标记的细胞,与损伤对照组相比,二者的左室结构与功能均明显改善(P<0.05);而心肌移植组对心功能的改善优于静脉移植组,二者的差异有统计学意义(P<0.05)。结论经静脉移植的MSCs能够在心肌局部分化成为类心肌细胞,并有效改善心脏功能;经心肌移植MSCs对心功能的改善更为有效。     

VEGF_(165)和Angiopoietin-1 cDNA治疗缺血心肌的实验研究

目的探讨血管内皮生长因子165(VEGF_(165))和血管生成素1(Angiopoietin-1,Ang1)基因导入对心肌缺血的治疗作用。方法分别构建编码VEGF_(165)和Ang1的真核表达载体。结扎SD大鼠冠状动脉前降支后1周,将缺血模型随机分为实验组(n=7)和对照组(n=7),在缺血区周围心肌内分别注射VEGF_(165)和Ang1的重组质粒和空质粒。1周后收取心脏,通过Langendorff心脏灌注模型实验,测定离体心脏的心功能。用免疫组织化学方法测定心肌注射区血管密度。结果正确构建了重组质粒pSecTag-VEGF和pSecTag-Ang1,并能在HEK293细胞中特异地表达出VEGF165和Ang1。同对照组相比,治疗后1周实验组左心室收缩压(LVSP)从(34.15±5.78)mmHg(1mmHg=0.133kPa)升高到(39.33±6.10)mmHg;左心室舒张末压(LVEDP)从(15.85±1.79)mmHg降低到(9.09±1.98)mmHg(P<0.05);左心室内压最大上升速率(+dp/dtmax)从(1413.52±417.73)mmHg/s升高到(1939.28±710.22)mmHg/s;左心室内压最大下降速率(-dp/dtmax)从(600.39±55.59)mmHg/s升高到(1030.53±401.22)mmHg/s(P<0.05);左心室做功(LVW)从(8213.88±303.68)mmHg/min升高到(15416.67±233.43)mmHg/min(P<0.05);冠状动脉流量(CF)从(3.2±0.31)mL/min升高到(4.09±0.80)mL/min(P<0.05),心肌注射区毛细血管数分别从(25±5)条/mm2增加到(46±8)条/mm2(P<0.05)。结论VEGF_(165)和Ang1基因导入能增强缺血心肌的血管新生和侧支循环的形成,改善心功能。     

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.     

骨髓间充质干细胞联合重组人生长激素治疗大鼠心肌坏死

目的:探讨骨髓间充质干细胞(MSCs)移植联合重组人生长激素(rhGH)皮下注射对大鼠心肌坏死模型坏死区血管新生的影响。方法:密度梯度离心和贴壁筛选法获得Wistar大鼠骨髓MSCs,液氮冷冻法制备大鼠左心室前壁心肌坏死模型,动物随机分为5组:假手术组(n=6),实验对照组(n=8),MSCs移植组(经BrdU标记的MSCs12．5×106个／50μl,分5点注射,n=8),rhGH注射组(rhGH 2 IU／kg·d,共14天,n=8)及MSCs+rhGH联合治疗组(n=8)。4周后处死动物,BrdU 免疫组化染色评价移植的MSCs向血管内皮细胞的生成、分化,苏木素伊红(HE)染色下评价坏死面积及血管密度。结果:各组间坏死面积未见统计学差异(P>0．05)。坏死区部分血管内皮细胞核呈BrdU阳性。MSCs移植组、rhGH 注射组以及MSCs+rhGH联合治疗组的血管密度明显高于实验对照组(P<0．001),而MSCs+rhGH联合治疗组坏死区的血管密度又高于MSCs移植组和rhGH注射组(P<0．05)。MSCs移植组与rhGH注射组之间坏死区的血管密度无统计学差异(P>0．05)。结论:心肌坏死急性期植入的MSCs能在坏死区存活、生长并可以向血管内皮细胞方向分化。MSCs移植可以增加坏死区血管密度,rhGH可以加强MSCs促血管生长的作用。     

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.     

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.     

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.     

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.