创面巨噬细胞分泌物对成纤维细胞的调节作用

采用体外培养体系研究家兔不同时期皮肤创面巨噬细胞上清液和冻融液对创面成纤维细胞增殖和胶原蛋白合成的调节作用。用皮下埋置自制不锈钢管法，直接获取手术后７、１４、２１、２８天活化的创面巨噬细胞，贴壁纯化后培养４８小时收集上清液；细胞经－３０℃与３７℃反复冻融３次收集冻融液。从兔皮肤３～５天刨面取新生肉芽组织，经组织培养获取创面成纤维细胞。测定成纤维细胞中３Ｈ－胸腺嘧啶和３Ｈ－脯氨酸的掺入量。结果上清液对成纤维细胞增殖和胶原蛋白合成具有不同程度的促进作用；冻融液主要表现为抑制作用。说明巨噬细胞能合成或分泌某些生物活性物质调节成纤维细胞的活动，从而控制创伤愈合的进程。     

PDGF-AB对成纤维细胞增殖及DNA合成的影响

目的：探讨ＰＤＧＦ－ ＡＢ促进伤口愈合及其在瘢痕增生中的作用机理。方法：取体外培养的人正常皮肤及增生性瘢痕成纤维细胞（ｎｏｒｍａｌｓｋｉｎｆｉｂｒｏｂｌａｓｔ,ＮｓＦｂ,ｈｙｐｅｒｔｒｏｐｈｉｃ ｓｃａｒｆｉｂｒｏｂｌａｓｔ,ＨＴｓＦｂ） ,用ＭＴＴ和３Ｈ－ ＴｄＲ掺入法观察ＰＤＧＦ－ ＡＢ对两种细胞增殖及ＤＮＡ合成的影响。结果：ＰＤＧＦ－ ＡＢ对两种成纤维细胞增殖及ＤＮＡ合成均有明显刺激作用,且均呈剂量依赖性关系,但作用有差异。结论：ＰＤＧＦ－ ＡＢ可能通过刺激成纤维细胞增殖及ＤＮＡ合成促进伤口愈合,但同时在瘢痕增生性疾病中可能起重要促进作用。     

鲑降钙素类似物[Glu~(31),Pr~o(32)-N(C_2H_5)_2]sCT的合成、二级结构及活性研究

通过侧链悬挂固相法合成了Ｃ端二乙胺基保护及用Ｇｌｕ取代Ｔｈｒ３１的鲑降钙素类似物［Ｇｌｕ３１，Ｐｒｏ３２－Ｎ（Ｃ２Ｈ５）２］ｓＣＴ，以降低Ｃ端酰胺基的降解，增加肽的稳定性。用圆二色谱（ＣＤ）法研究了肽［Ｇｌｕ３１，Ｐｒｏ３２－Ｎ（Ｃ２Ｈ５）２］ｓＣＴ在水及三氟乙醇（ＴＦＥ）中的二级结构。它在大鼠体内的降钙活性为５２７６．９ＩＵ／ｍｇ。     

鲑降钙素类似物[Glu^31,Pro^32—N（C2H5）2]sCT的合成,地级…

通过侧链悬挂固相法合成了Ｃ端二乙胺基保护及用Ｇｌｕ取代Ｔｈｒ＾３１的鲑降钙素类似物［Ｇｌｕ＾３１，Ｐｒｏ＾３２－Ｎ（Ｃ２Ｈ５）２］ｓＣＴ，以降低Ｃ端酰胺基的降解，增加肽的稳定性。用圆二色谱（ＣＤ）法研究了肽［Ｇｌｕ＾３１，Ｐｒｏ＾３２－Ｎ（Ｃ２Ｈ５）２］ｓＣＴ在水及三氟乙醇（ＴＦＥ）中的二级结构。它在大鼠体内的降钙活性为５２７６．９ＩＵ／ｍｇ。     

川芎嗪和硫氮唑酮对照射后NIH/3 T3成纤维细胞增殖及bFGF表达的影响

目的　探讨川芎嗪及硫氮唑酮对照射后ＮＩＨ／３Ｔ３ 成纤维细胞增殖及ｂＦＧＦ表达的影响,为防治放射性肺纤维化的药物研究提供实验依据。方法　采用ＭＴＴ 法测定离体培养的ＮＩＨ／３Ｔ３ 成纤维细胞增殖程度并用免疫细胞化学ＡＢＣ 方法观察ｂＦＧＦ蛋白表达改变。结果　不同组ＭＴＴ法测得光密度值,对照组为０－７１ ±０－２２ ;照射组（Ⅰ） 为１－０３ ±０－２８ ;川芎嗪＋ 照射组为０－４７±０－１５ ;照射组（ Ⅱ） 为１－０５ ±０－２１;硫氮唑酮＋ 照射组为２－０２ ±０－２１;不同组ｂＦＧＦ 蛋白表达平均光密度：对照组为０－０２８±０－００５ ;照射组为０－０４４ ±０－００６ ;川芎嗪＋ 照射组为０－０２９ ±０－００１ ;硫氮唑酮＋ 照射组为０－０３６ ±０－００３。结论　川芎嗪明显抑制γ射线照射后ＮＩＨ／３Ｔ３ 成纤维细胞的增殖及ｂＦＧＦ蛋白表达,硫氮唑酮则不具有这一作用。     

脑缺血再灌流时［~3H］－三磷酸肌醇放射活性及突触体游离Ca~（2＋）的变化

研究大鼠脑缺血再灌流时［３Ｈ］－ＩＰ３放射活性及突触体（Ｃａ２＋）ｉ的变化，结果示缺血１ｍｉｎ就启动了肌醇脂质信使系统，引起［３Ｈ］－ＩＰ３显著增高，缺血２０ｍｉｎ突触体（Ｃａ２＋）ｉ增高，且持续至再灌流７ｄ，磷脂酶Ｃ抑制剂ＰＭＳＦ治疗能显著地抑制突触体（Ｃａ２＋）ｉ的升高，减轻海马ＣＡ１区缺血性神经元损伤。     

PDGF-B基因表达及其对成纤维细胞增生和胶原合成的促进作用

目的建立稳定表达人血小板衍生生长因子－ＢＢ（ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ－ＢＢ，ＰＤＧＦ－ＢＢ）的细胞株，并考察该重组蛋白与损伤修复有关的生物学作用。方法将含ＰＤＧＦ－Ｂ基因的真核表达载体ｐｃＤＮＡ３转染小鼠成纤维细胞ＮＩＨ３Ｔ３中，在Ｇ４１８选择压力下得到抗性克隆，收集扩增的抗性克隆培养液并制备细胞膜上ＰＤＧＦ－ＢＢ蛋白质，３Ｈ－ＴｄＲ掺入法表达产物的生物学活性，并以３Ｈ－Ｐｒｏ掺入测胶原合成率。结果免疫荧光染色法证实抗性细胞中ＰＤＧＦ－ＢＢ的表达，膜上ＰＤＧＦ－ＢＢ显著促进ＮＩＨ３Ｔ３细胞ＤＮＡ合成，活性可达约３１．２Ｕ／１０６细胞，而培养液的致丝裂活性低于可检测水平。抗性细胞的胶原合成率显著增高。结论ＰＤＧＦ－ＢＢ的真核表达系统已得以成功构建，该重组蛋白可促进成纤维细胞增生和胶原合成，提示其在损伤修复中有潜在应用价值     

异基因骨髓移植供者使用粒细胞集落刺激因子对骨髓重建与移植物抗宿主病的影响

异基因外周血干细胞移植，具有造血重建快，粒细胞缺乏和血小板减少期缩短，增加了移植期的安全性，由于输入Ｔ淋巴细胞数量较多，可能增加移植物抗宿主病（ｇｒａｆｔｖｅｒｓｕｓｈｏｓｔｄｉｓｅａｓｅ，ＧＶＨＤ）的发病率，特别是慢性ＧＶＨＤ［１］。在小鼠不相合的骨髓移植实验研究，提前使用粒细胞集落刺激因子（Ｇ－ＣＳＦ）动员供者骨髓，移植后降低了重症ＧＶＨＤ的发生，提高了生存率［２］。在自体骨髓移植中，加用少量外周血干细胞，可加速造血重建［３］。本文在ＨＬＡ相合的异基因骨髓移植中，提前给供者使用（Ｇ－ＣＳＦ）…     

氧化修饰高密度脂蛋白对鼠腹腔巨噬细胞增殖的影响

已有证据表明,巨噬细胞源性泡沫细胞与血管平滑肌细胞相似,在人动脉粥样硬化（ＡＳ）斑块原位呈增殖现象,提示巨噬细胞源性泡沫细胞增殖在ＡＳ发展中起重要作用［１,２］。最近,国外发现［３,４］氧化低密度脂蛋白（Ｏｘ－ＬＤＬ）可以刺激巨噬细胞增殖,这一发现为氧化修饰脂蛋白致Ａｓ机制提供了新线索,目前国内未见有关报道。鉴于高密度脂蛋白（ＨＤＬ）氧化修饰后抗Ａｓ能力减弱,并具部分致ＡＳ效应。本研究观察比较了氧化ＨＤＬ（Ｏｘ－ＨＤＬ）和Ｏｘ～ＬＤＬ对鼠腹腔巨噬细胞增殖的影响,以期进一步阐明Ｏｘ－ＨＤＬ在Ａｓ中…     

吡啶-2,6(1H,3H)二酮生物碱对血小板聚集及血栓素B_2和6-酮-前列腺素_(Iα)的影响

观测ＳＨ１对ＡＤＰ，ＡＡ，Ｃｏｌａｇｅｎ诱导的兔血小板聚集及ＴＸＢ２和６－ｋｅＴＯ－ＰＧＦＩα生成的影响。用比浊法测定了透骨草提取的单体生物碱结晶ＳＨ１体外对兔血小板聚集的影响，用放免法测定ＴＸＢ２和６－ｋｅＴＯ－ＰＧＦＩα的含量。结果：ＳＨ１０．８～３．０ｍｍｏｌ／Ｌ范围内明显抑制ＡＡ，ＡＤＰ，Ｃｏｌａｇｅｎ诱导的兔血小板聚集。ＳＨ１对三种诱导剂的最大抑制率分别为６２．１６％，４５．２５％，５３．６７％。大剂量组明显加快ＡＤＰ诱导的血小板聚集后的解聚速度，ＳＨ１显著延长Ｃｏｌａｇｅｎ的诱导起聚时间。其抑制作用有明显的量效关系，对ＡＡ诱导的ＴＸＢ２产生明显抑制作用，可使６－ｋｅＴＯ－ＰＧＦＩα的含量略有增加，但６－ｋｅＴＯ－ＰＧＦＩα／ＴＸＢ２比值显著增大     

The effect of estrogen on ovine anterior cruciate ligament fibroblasts: cell proliferation and collagen synthesis

Estrogen has been implicated as a causal factor for anterior cruciate ligament injuries in women. Studies have demonstrated a decrease in anterior cruciate ligament fibroblast proliferation and collagen synthesis at supraphysiologic levels of estrogen in a rabbit model. HYPOTHESIS: The authors hypothesized that physiologic levels of estrogen would have no significant effect on anterior cruciate ligament fibroblast proliferation and collagen synthesis in an ovine model. METHODS: Anterior cruciate ligament fibroblasts were isolated from sheep knees using routine cell culture methods. The cells were exposed to 17beta-estradiol at physiologic concentrations of 2.2, 5, 15, 25, 250, and 2500 pg/ml. Cell proliferation was determined by cell counts on days 4 and 6. Collagen synthesis was determined by (3)H-proline incorporation on day 4. Immunohistochemistry was performed to detect estrogen receptors. RESULTS: Immunohistochemistry demonstrated the presence of estrogen receptors in ovine anterior cruciate ligament fibroblasts. There was no significant difference in anterior cruciate ligament fibroblast proliferation or collagen synthesis regardless of 17beta-estradiol concentration. CONCLUSIONS: Based on results of this study, and given the low turnover of collagen in ligaments, it is unlikely that a 2- to 3-day per month increase in circulating estrogen would result in rapid, clinically significant alterations in material properties of the anterior cruciate ligament in vivo. The etiology of noncontact anterior cruciate ligament injuries is complex and multifactorial in nature, meriting further investigation.     

放射性肺损伤早期肺组织重建机制的探讨

目的 探讨Ⅳ型胶原和MMP-9在放射性肺损伤早期肺组织重建中的作用。方法 用噻唑蓝(MTT)检测1～10 Gy ⁶⁰Co γ 射线照射对人肺成纤维细胞(Fb)增殖的影响;用酶联免疫吸附法测定经5和7 Gy照射后Fb中Ⅳ型胶原和MMP-9的变化;从经25 Gy照射后大鼠肺泡灌洗液中分离巨噬细胞,制备条件培养液(CMAM)并刺激肺Fb,用MTT检测对细胞增殖的影响;同时用ELISA测定对Fb合成Ⅳ型胶原和MMP-9的影响;于照射后不同时间取大鼠肺组织进行Ⅳ型胶原和MMP-9免疫组化染色。结果 1～7 Gy照射能促进肺Fb增生;5和7 Gy照射能促进Fb合成MMP-9,但不能促进Ⅳ型胶原合成;CMAM不但能促进Fb增生和MMP-9合成,也能合成和释放Ⅳ型胶原;受照后1周大鼠肺组织出现Ⅳ型胶原沉积。结论 照射能直接刺激肺Fb增生,但不能产生Ⅳ型胶原;Ⅳ型胶原的合成与照射后肺内巨噬细胞和Fb的相互作用有关,并可导致肺损伤重建的发生。     

Prostaglandin E2 affects proliferation and collagen synthesis by human patellar tendon fibroblasts.

OBJECTIVE: To determine the effect of prostaglandin E2 on proliferation and collagen synthesis by human patellar tendon fibroblasts. DESIGN AND SETTING: Controlled laboratory study. METHODS: Human patellar tendon fibroblasts were treated with different concentrations (1, 10, 100 ng/mL) of prostaglandin E2 in cultures. Fibroblasts without prostaglandin E2 treatment were used as the control group. The fibroblast proliferation and collagen synthesis were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and Sircol collagen assay, respectively. MAIN OUTCOME MEASURED: Changes in proliferation and collagen production of human patellar tendon fibroblasts. RESULTS:: At 1 ng/mL of prostaglandin E2, there was no significant effect on fibroblast proliferation compared with the control group. At concentrations of 10 ng/mL and 100 ng/mL prostaglandin E2, however, fibroblast proliferation significantly decreased, by 7.3% (P = 0.002) and 10.8% (P < 0.0001), respectively, compared with the control group. At 1 ng/mL of prostaglandin E2, collagen production of the tendon fibroblasts was unaffected. However, at both 10 ng/mL and 100 ng/mL prostaglandin E2, collagen production was significantly decreased, by 45.2% (P < 0.0001) and 45.7% (P < 0.0001), respectively, compared with the control group. The levels of collagen production between these 2 dosages did not differ significantly. CONCLUSIONS: Prostaglandin E2 affects the proliferation of and collagen production by human patellar tendon fibroblasts in a dosage-dependent manner. CLINICAL RELEVANCE: Based on these in vitro findings, we speculate that production of prostaglandin E2 in tendons might play some role in the acellularity and matrix disorganization seen in exercise-induced tendinopathy.     

The effect of ciprofloxacin on tendon, paratenon, and capsular fibroblast metabolism

The pathologic mechanisms underlying fluoroquinolone-induced tendinopathy are poorly understood. The observed incidence of tendinitis and tendon rupture in patients treated with ciprofloxacin hydrochloride suggests that the fluoroquinolone antibiotics alter tendon fibroblast metabolism. The purpose of this study was to examine the effect of ciprofloxacin on fibroblast metabolism in vitro. Canine Achilles tendon, paratenon, and shoulder capsule specimens were maintained in culture with ciprofloxacin (5, 10, or 50 microg/ml). Fibroblast proliferation, collagen synthesis, proteoglycan synthesis, and matrix-degrading activity were analyzed. Incubation of Achilles tendon, Achilles paratenon, and shoulder capsule fibroblasts with ciprofloxacin resulted in a statistically significant 66% to 68% decrease in cell proliferation compared with control cells at day 3 in culture. Ciprofloxacin caused a statistically significant 36% to 48% decrease in collagen synthesis compared with controls in all fibroblast cultures. Ciprofloxacin caused a statistically significant 14% to 60% decrease in proteoglycan synthesis in all fibroblast cell lines. Compared with unstimulated control fibroblasts, culture media from Achilles tendon, paratenon, and shoulder capsule cells that were exposed to ciprofloxacin demonstrated statistically significant increases in matrix-degrading proteolytic activity after 72 hours in culture. This study demonstrates that ciprofloxacin stimulates matrix-degrading protease activity from fibroblasts and that it exerts an inhibitory effect on fibroblast metabolism. The increase in protease activity and the inhibition of both cell proliferation and the synthesis of matrix ground substance may contribute to the clinically described tendinopathies associated with ciprofloxacin therapy.     

细胞内钙调控对心肌成纤维细胞增殖的作用

目的:探讨不同来源的细胞内Ca2+([Ca2+]i)对心肌成纤维细胞(fibroblasts,PBs)丝裂素活化蛋白激酶(MAPK)介导的增殖反应的作用。方法:以培养的大鼠FBs为模型,用血管紧张素Ⅱ(AngⅡ)刺激FBs细胞外Ca2+跨膜内流、三磷酸肌醇(IP3)刺激胞内Ca2+释放,应用钙荧光指剂Fura-2/AM动态观测心肌细胞[ca2+]i浓度,γ-32P-ATP掺入法和免疫印迹(westen blot)测MAPK活性及蛋白含量,氚-亮氨酸(3H-Leu)、氚-胸腺嘧定(3H-TdR)掺入量作为FBs增殖的指标。结果:AngⅡ、IP3均能显著增加FBs[Ca2+]j浓度、MAPK活性及蛋白含量,并提高3H-Leu、3H-TdR掺入量,与对照组FBs相比差异显著,P<0.01。结论:激活[Ca2+]i使MAPK活性及含量明显增加而促进FBs的增殖,FBs的增殖与[ca2+]i浓度增加有关,与[Ca2+]i的来源无关。     

Basis of FLT as a cell proliferation marker: comparative uptake studies with [3H]thymidine and [3H]arabinothymidine, and cell-analysis in 22 asynchronously growing tumor cell lines

The usefulness of radiolabeled 3'-fluoro-3'-deoxythymidine (FLT), a thymidine derivative with affinity to cytoplasmic thymidine kinase 1 (TK1), as a tumor proliferation marker was evaluated using [3H]FLT and 22 cultured tumor cell lines. Asynchronously growing tumor cells were used for studies to mimic in vivo status of tumors. FLT uptake in each cell line was compared with [3H]thymidine ([3H]Thd) uptake and %S-phase fraction, both known as acceptable markers of proliferation. Uptake of the mitochondrial TK2 specific substrate [3H]arabinothymidine ([3H]AraT) was studied as a reference. Metabolic fate of FLT in tumor cells was also analyzed to elucidate the retention mechanism of FLT. [3H]FLT uptake was mildly correlated with the %S-phase fraction (r=0.76, p<0.0001) and correlated better with [3H]Thd uptake (r=0.88, p<0.0001). In contrast, the TK(2) specific substrate, [3H]AraT, was not significantly correlated with the %S-phase fraction (r=0.19, p=0.39), although it showed some correlation with the [3H]Thd uptake (r=0.47, p<0.05). Over 90% of radioactivity of [3H]Thd was found in the DNA fraction after 60 minutes incubation. In contrast, most of the radioactivity of [3H]FLT was found in the acid-soluble fraction (95%). [3H]FLT incorporation into the DNA fraction was negligible (0.2%). The [3H]AraT was mainly distributed in the acid-soluble fraction (70%) and the DNA fraction (20%). From our results, we concluded that FLT uptake in tumor cells reflects tumor cell proliferation. However, much more convincing validation is needed to clarify the difference between FLT and true substrates for DNA synthesis, like thymidine.     

Feasibility studies of 4'-[methyl-(11)C]thiothymidine as a tumor proliferation imaging agent in mice

This study reports on the radiosynthesis and feasibility studies of 4'-[methyl-(11)C]thiothymidine ([methyl-(11)C]S-dThd) as a tumor proliferation imaging agent. [Methyl-(11)C]S-dThd was synthesized by rapid methylation of corresponding 5-trimethylstannyl- or 5-tributylstannyl-precursor via a palladium-promoted Stille cross-coupling reaction with [(11)C]methyl iodide. The decay-corrected radiochemical yields of [methyl-(11)C]S-dThd synthesized by the corresponding 5-trimethylstannyl-precursor and 5-tributylstannyl-precursor based on [(11)C]CO(2) were 18.9% and 14.5%, respectively. The radiochemical purity of [methyl-(11)C]S-dThd was always greater than 99%. The specific activities of [methyl-(11)C]S-dThd synthesized by the corresponding 5-trimethylstannyl-precursor and 5-tributylstannyl-precursor were 47 GBq/mumol and 121 GBq/mumol, respectively, at the end of the synthesis. The total synthesis time was 30 min after the end of bombardment. The comparison between in vivo distribution of [methyl-(14)C]S-dThd and that of [methyl-(3)H]FLT showed that tracer uptake was comparable in nonproliferating tissues. In contrast, [methyl-(14)C]S-dThd showed significantly higher uptake in proliferating tissues than did [methyl-(3)H]FLT. [Methyl-(11)C]S-dThd uptake levels in five different tumor tissues were well correlated with the DNA synthesis levels determined by [2-(14)C]thymidine DNA incorporation. At 30 min after injection, plasma analysis found 95% of the activity in unmetabolized form. The microPET imaging of the C6 glioma xenograft showed significantly high uptake in the tumor and urinary bladder, followed by the intestine and marrow. Our results demonstrated that the tumor uptake of [methyl-(11)C]S-dThd was higher than that of [methyl-(3)H]FLT and was well correlated with the DNA synthesis level. Consequently, 4'-[methyl-(11)C]thiothymidine has promise for the imaging of tumor cell proliferation by positron emission tomography.     

Autologous chondrocyte implantation: cells phenotype and proliferation analysis

The phenotype and proliferation of human chondrocytes in culture were analyzed before they were implanted as autologous graft in cartilage lesions. During ten autologous chondrocyte implantations in articular cartilage lesions of the knee in ten patients, small amounts of cells to be implanted were collected and analyzed by morphology, cytochemistry (alcian blue, safranin-O), and immunocytochemistry (antibodies anti-S100 protein, anti-collagen types I and II, anti-chondroitin-S). In four cases the cells were also cultured for 3 weeks. At 1, 10, and 20 days of culture cells were incubated with 1 microCi/ml [3H]thymidine for proliferation analysis. In all cases the cells showed the morphological appearance of mature chondrocytes, stained positively for alcian blue and safranin-O, and revealed a strong immunoreaction for S-100 protein, type II collagen, and chondroitin-S but not for type I collagen. Radioisotope assay of chondrocyte proliferation at 1, 10, and 20 days of culture revealed a progressive increase in [3H]thymidine incorporation. These findings indicate that the cells before autologous implantation maintain their differentiated phenotype of mature chondrocytes and proliferate greatly. This analysis is an essential step preceding wider use of this treatment in humans. However, other biological aspects of the autologous chondrocyte graft remain to be elucidated.     

hBMP7基因转染对兔骨髓间充质干细胞增殖及胶原合成的影响

使用含hBMP7基因的重组逆转录病毒液感染兔骨髓间充质干细胞（BMSc）,MTT法检测细胞增殖能力,流式细胞仪检测细胞周期,^3H脯氨酸掺故法检测I型胶原合成和表达情况。结果显示,经hBMP7基因转染的BMSc与空载体转染及未转染的BMSc在细胞增殖、细胞周期表现方面无显著性差异,经转染的BMSc合成胶原显著高于空载体转染和未转染者。提示采用逆转录病毒介导转染BMSc后能促进细胞的胶原合成,对BMSc增殖和细胞周期无显著影响,有利于进一步 应用于构建组织工程化骨组织。