1,25（OH）2维生素D3对人树突状细胞成熟及其介导免疫耐受的影响

本研究旨在探讨1,25(OH)2维生素D3〔1,25(OH)2Vit D3〕对人树突状细胞(DC)分化、成熟及功能的影响及其机制。在体外将人外周血单个核细胞诱导分化成DC,实验组加入1,25(OH)2Vit D31 nmol/L培养9 d,对照组加入等量无水乙醇,流式细胞仪检测DC表面共刺激因子表达水平。混合淋巴细胞培养后,用MTT法评估DC刺激同种异体T细胞增殖的能力。Western blot检测DC吲哚胺2,3-双加氧酶(IDO)蛋白表达。结果表明,与对照组相比,实验组DC表面标志CD80、CD83、CD86表达率低于对照组(P<0.05),CD1a高于对照组(P<0.05);CD80、CD83、CD86、CD1a表达率分别为(40.43±9.83)%、(20.04±4.73)%、(14.45±5.38)%,(58.48±10.72)%;对照组CD80、CD83、CD86、CD1a表达率分别为(29.36±13.34)%、(35.91±10.19)%、(27.15±11.64)、(72.20±12.79)%。实验组DC刺激同种异体T细胞增殖的能力受到抑制;DC表达IDO蛋白上调。结论:1,25(OH)2Vit D3抑制DC的成熟,通过上调DC的IDO蛋白表达抑制DC刺激同种异体T淋巴细胞增殖及介导免疫耐受。     

活性维生素D3调控肾小球系膜细胞增殖与凋亡机制研究

作为一种类固醇激素,活性维生素D3（1,25（0H）1D3）可抑制肾小球系膜细胞的增殖,而细胞的增殖又与mTOR信号传导通路相关,本文就1,25（OH）2D3通过mTOR信号传导通路调控肾小球系膜细胞增殖与凋亡的机制作一综述。     

青藤碱对大鼠肾小球系膜细胞增殖和凋亡的影响

目的探讨青藤碱(sinomenine,SIN)对大鼠肾小球系膜细胞(mesangial cell,MC)增殖和凋亡的影响。方法体外培养的大鼠MC,分为SIN分为高、中、低剂量组和空白对照组。采用四甲基偶氮唑蓝(MTT)法检测细胞的增殖情况,碘化丙啶染色后流式细胞术检测凋亡率,吖啶橙/溴乙锭染色后采用荧光显微镜观察细胞凋亡水平。结果MTT实验和流式细胞检测结果显示,与空白对照组比较,SIN高(P<0.01)、中(P<0.05)、低(P<0.05)剂量组MC增殖降低,凋亡增加,吖啶橙/溴乙锭染色结果显示SIN干预的MC出现凋亡的百分率增加。结论青藤碱可抑制大鼠肾小球系膜细胞增殖并促进其凋亡,可能会使肾脏病理性损害减轻。     

青蒿琥酯对脂多糖诱导的大鼠肾小球系膜细胞凋亡的影响

目的:观察青蒿琥酯对脂多糖诱导的大鼠肾小球系膜细胞(glomerular mesangial cells,GMC)凋亡的影响。方法:用不同浓度的青蒿琥酯刺激脂多糖诱导增殖的大鼠肾小球系膜细胞,HE染色观察凋亡细胞形态,DAPI荧光染色观察细胞核凋亡情况,用流式细胞仪检测细胞凋亡率。结果:青蒿琥酯对系膜细胞凋亡有明显诱导作用,呈一定剂量依赖性。结论:青蒿琥酯能够剂量依赖性地诱导大鼠系膜细胞凋亡。     

表皮生长因子对大鼠肾小球系膜细胞增殖的影响

目的:探讨表皮生长因子对体外肾小球系膜细胞增殖作用的影响,为进一步研究系膜细胞增殖的发生机制提供依据。方法:通过肾小球系膜细胞体外培养技术,用含有不同浓度表皮生长因子的培养液培养系膜细胞,作用不同时间,以噻唑蓝法检测细胞增殖能力,观察不同浓度、作用时间下系膜细胞增殖情况。结果:表皮生长因子在0～100ng/mL浓度范围内,其促增殖作用随浓度的增加而增大,呈剂量依赖性(P<0.01),当浓度持续增高时,其促增殖能力有所降低(P<0.05)。加入10ng/mL表皮生长因子作用12～24h促增殖能力逐渐提高,24h达到高峰(P<0.05),随后又随着时间的延长而逐渐降低(P<0.05)。结论:表皮生长因子在一定浓度和时间范围内具有促进肾小球系膜细胞增殖的作用。     

大黄酸可影响高糖培养SD大鼠肾小球系膜细胞的凋亡

背景：含有大黄的复方中药消可宁能有效防治早期糖尿病肾病。目的：观察大黄酸对髙糖培养的SD大鼠肾小球系膜细胞凋亡的影响。方法：以20,40,80μmol/L浓度的大黄酸刺激髙糖培养的肾小球系膜细胞,苏木精-伊红染色观察凋亡细胞形态,DAPI荧光染色观察细胞核凋亡情况,流式细胞仪观察细胞凋亡率。结果与结论：苏木精-伊红染色及DAPI染色结果显示大黄酸可促进髙糖培养的肾小球系膜细胞的凋亡,且与浓度与时间成正相关。各组肾小球系膜细胞的细胞凋亡率差异有显著性意义（P〈0.05）,表明大黄酸对髙糖培养的肾小球系膜细胞凋亡产生影响且与浓度及时间成正相关。     

1,25(OH)_2D_3联合钙剂对应用不同剂量糖皮质激素治疗的原发性肾病患者骨密度的影响

目的观察1,25(OH)2D3和钙剂联合治疗对使用不同剂量糖皮质激素治疗的原发性肾脏疾病患者骨密度的影响。方法将58位应用糖皮质激素治疗的原发性患者先按使用激素的剂量分为小剂量组(A组)和大剂量组(B组),每组内随机分为2组:治疗组予1,25(OH)2D30.25μg/d和碳酸钙600mg/d联合治疗;对照组单独使用碳酸钙600mg/d治疗。观察治疗前及治疗12周后腰椎及股骨颈骨密度,同时检测血清甲状旁腺素、血钙、血磷、血碱性磷酸酶等指标。结果A、B2组中治疗组与对照组治疗前后血清PTH、血钙、血磷、血AKP水平比较均无明显差异;对照组治疗12周后的腰椎及股骨颈骨密度均较治疗前明显下降(P0.05)。A组患者中,治疗12周后治疗组患者的BMD水平高于对照组(P0.05),且与治疗前无明显差异;B组患者中,治疗12周后治疗组患者的BMD水平与对照组无明显差异。结论对于使用中小剂量激素的原发性肾脏疾病患者,1,25(OH)2D3和钙剂联合治疗可预防其骨量减少。     

大黄素干预高糖培养大鼠肾小球系膜细胞的凋亡

背景:前期工作已经证实中药复方消可宁(制大黄、制附子、生黄芪)能有效防治早期糖尿病肾病并获得国家专利(专利号:200410064899X)。目的:观察大黄主要活性成分大黄素对高糖培养的SD大鼠肾小球系膜细胞凋亡的影响。方法:用20,40,80μmol/L大黄素刺激高糖培养的SD大鼠肾小球系膜细胞,苏木精-伊红染色观察凋亡细胞形态,4’,6-二脒基-2-苯基吲哚荧光染色观察细胞核凋亡情况,流式细胞仪观察细胞凋亡率。结果与结论:苏木精-伊红染色及4’,6-二脒基-2-苯基吲哚染色结果显示大黄素对高糖培养的肾小球系膜细胞的凋亡有影响,且与浓度与时间成正相关,细胞凋亡率组间差异有显著性意义(P<0.05)。提示大黄素可诱导肾小球系膜细胞凋亡,且与药物浓度及时间成正相关。     

肝细胞生长素在体外对大鼠肾小球系膜细胞生物活性的影响

目的 观察低分子肝细胞生长素(HPN)对炎症因子刺激下的大鼠肾小球系膜细胞增殖及产生TGFβ的影响。方法 用^3H—TdR掺入法检测肾小球系膜细胞(GMC)增殖变化，用ELISA法检测肾小球系膜细胞培养上清中TGFβ的水平。结果 在无IL-1β存在时，浓度为400u/孔的低分子肝细胞生长素对常规培养的系膜细胞增殖有明显抑制作用(P＜0．01)，而对TGFβ的产生无影响；但它对IL-β刺激下的大鼠系膜细胞增殖和TGFβ产生均有明显抑制作用(均P＜0．01)。低分子肝细胞生长素在100u/孔浓度时无上述作用。结论 高剂量HPN可抑制体外培养的大鼠系膜细胞增殖，降低IL-β刺激系膜细胞产生TGFβ。低分子肝细胞生长素无种属特异性和组织特异性。     

高糖对大鼠肾系膜细胞Smad2/3及Smad7表达的影响

目的:观察不同浓度高糖刺激后大鼠肾小球系膜细胞内Smad2/3和Smad7蛋白的表达,探讨糖尿病时肾脏Smad信号通路的改变。方法:将体外培养的大鼠肾系膜细胞分别设正常对照组(葡萄糖浓度5.6mmol/L)、20mmol/L高糖组、30mmol/L高糖组、甘露醇组。用细胞免疫荧光染色法及激光共聚焦显微镜检测各组细胞Smad2/3及Smad7蛋白的表达。结果:正常对照组系膜细胞Smad2/3蛋白表达较弱,Smad7蛋白表达较强。两高糖组Smad2/3表达较正常对照组强(P﹤0.05),呈浓度依赖性;Smad7表达较正常对照组弱(P﹤0.05),呈浓度依赖性。甘露醇组与正常对照组无明显差异(P﹥0.05)。结论:高糖可诱导肾系膜细胞Smad2/3蛋白表达增强,Smad7蛋白表达减弱。提示Smad信号通路参与了糖尿病肾病的发病机制。     

Trans- and paracellular calcium transport across the colonic mucosa after short- and long-term treatment with 1,25-dihydroxyvitamin D3

The electrical parameters and the unidirectional fluxes of 45Ca and 3H-mannitol were measured in preparations of rat colon descendens freed from the muscularis externa and mounted in a modified Ussing-chamber. Two criteria were used to differentiate between changes in the trans- and the paracellular calcium transport after treatment with 1,25(OH)2D3: the fluxes of the simultaneously measured 3H-mannitol as a paracellular marker; the 45Ca fluxes in preparations with clamped potentials. After a short-time (6 h) pretreatment by s.c. administration of 1,25(OH)2D3 (250 ng kg-1) in normal rats the mucosa (m) to serosa (s) 45Ca flux under short circuit conditions increased about 65%, whereas the electrical parameters and the 3H-mannitol fluxes remained unchanged. In clamped epithelia the PD-independent m to s 45Ca flux was increased, whereas the PD-dependent flux remained unchanged. In contrast, after long-time (4 days) induction by 1,25(OH)2D3 the m to s 45Ca flux increased under short circuit conditions by about 100% and the m to s 3H-mannitol flux increased by 50%, PD and Isc decreased by more than 60%, whereas tissue resistance was the same, in clamped epithelia the calculated PD-independent, transcellular m to s 45Ca flux was 2.4 times and the PD-dependent, paracellular 45Ca-flux was 1.9 times higher than in controls, whereas the s to m 45Ca flux remained unchanged. On the basis of the relevant references the following conclusions were drawn: after short-time exposure to 1,25(OH)2D3 only the PD-dependent, transcellular m to s calcium transport is increased; this is probably due to a liponomic effect of 1,25(OH)2D3 at the brush border membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of bone marrow cells from myelodysplastic patients in the presence of 1,25 dihydroxyvitamin D3 or 13-cis retinoic acid

The separate effects of vitamin D3 (1,25(OH)2D3) and 13-cis retinoic acid on the differentiation in liquid culture of marrow cells from seven patients with myelodysplastic syndrome (MDS) were studied. Following incubation with 1,25(OH)2D3, an increasing number of myeloid cells acquired the morphological appearance of mature monocyte-macrophages and reacted positively to fluoride-sensitive naphthyl acetate esterase and specifically bound My4 monoclonal antibody (McAb). Incubation of bone marrow cells with 13-cis retinoic acid enhanced the number of cells with the morphological appearance of metamyelocytes and mature granulocytes as well as those that reacted positively with AS-D naphthol chloroacetate esterase. The results suggest that the differentiation pattern of myeloid precursor cells from MDS patients can be modulated by 1,25(OH)2D3 and 13-cis retinoic acid.     

Transport of vitamin D sterols in human plasma: effect of excess vitamin D, 25 hydroxyvitamin D and 1,25 dihydroxyvitamin D

Abstract. Vitamin D and its more active metabolites, 25 hydroxyvitamin D (25-OH-D) and 1,25-dihydroxy-vitamin D (1,25-(OH)₂-D), are transported in human plasma on a specific binding protein (DBP), which has been shown to have an α-globulin electrophoretic mobility. Since the concentration of DBP in normal human plasma is approximately 5 μmol/l, whereas that of all the vitamin D metabolites is less than 0·2 μmol/l, DBP is less than 3% saturated under physiological conditions. We have studied the transport of the above-mentioned metabolites in human plasma in vitro at normal and saturating concentrations. Human plasma was incubated with increasing amounts of vitamin D metabolites together with their radiolabelled tracers. Ultracentrifugation was used to isolate plasma lipoproteins (density, d < 1·21 g/ml) and agarose gel electrophoresis of lipoprotein-free plasma (d > 1·21 g/ml) to separate DBP (α globulin) from albumin. The recovery of the tracer in plasma proteins was always more than 80%. At physiological concentrations [³H]25-OH-D bound almost exclusively to DBP (98%), [³H]vitamin D or [¹⁴C]vitamin D bound both to DBP and to lipoproteins (40%), and [³H]1,25-(OH)₂-D bound to DBP (62%), to lipoproteins (15%) and also to albumin (23%). When the concentration of vitamin D metabolites was increased, DBP became saturated. The binding capacity of DBP was similar for all three sterols, about 5 μmol/l plasma, or one mole of sterol per mole of protein, but the saturating concentration was different for the three sterols (vitamin D > 1,25-(OH)₂-D > 25-OH-D). 25-OH-D had the greatest affinity for DBP, and it completely displaced both vitamin D and 1,25(OH)₂-D from DBP at higher concentrations. All sterols bound to both plasma lipoproteins and albumin: vitamin D preferentially to lipoproteins and both 25-OH-D and 1,25-(OH)₂-D to albumin. A similar binding pattern for vitamin D in plasma was observed previously by us in a child with vitamin D toxicity. The increased binding of vitamin D to lipoproteins and especially to albumin may help explain the pathogenesis of toxicity in hypervitaminosis D, where the plasma levels of the more active metabolites are insufficient to account for the clinical signs.     

1,25二羟维生素D3对哮喘小鼠的Rac1-IL33-ILC2通路的作用

目的观察1,25二羟维生素D31,25-(OH)2-D3对哮喘小鼠Ras相关的C3肉毒素底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)、白细胞介素-33(IL-33)和2型固有淋巴细胞(type 2 innate lymphocytes,ILC2)的影响。方法将30只7周龄BALB/c雌性小鼠随机分为对照组、哮喘组、干预组,每组各10只小鼠,卵清蛋白混合液致敏并雾化吸入建立哮喘小鼠模型,干预组每次激发前腹腔注射1,25(OH)2-D3混合液0.08 ml,对照组和哮喘组以生理盐水代替。末次激发24 h后对小鼠肺组织中IL-33、Rac1、ILC2进行检测并比较。结果与对照组比较,哮喘组Rac1下降,IL-33水平增高,ILC2数目升高。且Rac1与IL-33、ILC2呈明显的负相关性(r=-0.954、r=-0.957,P<0.05)。干预组IL-33及ILC2数目较哮喘组显著下降,Rac1较哮喘组显著上升,差异有统计学意义(P<0.05)。结论哮喘小鼠存在Rac1下降,IL-33水平增高,ILC2数目升高的免疫变化;1,25-(OH)2-D3可能通过上调哮喘小鼠Rac1的表达,导致IL-33等细胞因子水平产生减少,进而抑制ILC2的活化、减轻哮喘的免疫炎症。     

Effect of 1,25-dihydroxyvitamin D3 on mesangial cell proliferation

We have studied the effect of 1,25-dihydroxyvitamin D3 (1,25, (OH)2D3) on mesangial cell growth. Previous studies have shown that the monocyte-macrophage is the principal effector cell in immune-mediated nephritis; this cell infiltrates the glomerular mesangium, and its products may have important effects on the physiology of the mesangial cell. One of the substances produced by the activated macrophage is 1,25,(OH)2D3. We have investigated the effect of 1,25,(OH)2D3 on mesangial cell growth and found that this vitamin D metabolite suppresses the proliferation of mouse mesangial cells as assessed by mesangial cell tritiated thymidine uptake and by cell counts; this substance also antagonizes the mitogenic effect of epidermal growth factor on mesangial cell growth. By comparison, the vitamin D metabolite 25 hydroxyvitamin D3 has no significant suppressive effect on the proliferation of mesangial cells. It has also been possible to demonstrate that 1,25,(OH)2D3 could suppress the growth of mesangial cells that had been committed to proliferate by the prior addition of epidermal growth factor. The results of these studies are relevant to our understanding of the pathogenesis of the cellular abnormalities that occur in immune-mediated nephritis, and especially in subjects who have concurrent hypertension, because a segment of subjects with hypertension have demonstrable abnormalities in the levels of circulating 1,25,(OH)2D3.     

糖尿病肾病患者血清1，25-二羟维生素D3水平及临床意义

目的探讨血清1,25-二羟维生素D。水平在糖尿病肾病（diabeticnephropathy,DN）进展中的作用。方法131例2型糖尿病患者分为单纯糖尿病组（DM组）30例、早期DN组（EDN组）32例、临床DN组（CDN组）35例与DN终末期组（ESDN组）34例,选择体检健康者30例为对照组,各组采用ELISA法检测血清1,25-二羟维生素D3和25-羟维生素D。水平。结果糖尿病及DN各组血清1,25-二羟维生素D3及25-羟维生素D。水平均明显低于对照组（P〈0．01）,且随DN逐渐进展二者水平均逐渐降低（P〈0．05）;糖尿病患者血清1,25-二羟维生素D3水平与尿微量白蛋白排泄率呈负相关（r=0．452,P=0．034）,与肾小球滤过率呈正相关（r=0．390,P=0．006）。结论血清1,25-二羟维生素D3水平检测可能有利于DN的早期发现和病情判断。     

1,25-二羟维生素D3治疗对早期糖尿病肾病相关炎性因子的影响

目的探讨早期糖尿病肾病(diabetic nephropathy,DN)患者体内1,25-二羟维生素D3与血清炎性因子水平的变化,观察1,25-二羟维生素D3对早期DN患者相关炎性因子的影响。方法 2型糖尿病患者238例中无蛋白尿者100例为DM组,初诊早期DN者138例为DN组,DN组再随机分为治疗组和对照组各69例。在血糖、血压稳定1周后治疗组给予常规治疗+骨化三醇胶丸,对照组给予常规治疗,2组疗程均为3个月。比较各组患者1,25-二羟维生素D3、24h尿蛋白定量及相关炎性因子的变化。结果治疗前DN组1,25-二羟维生素D3水平低于DM组(P<0.05),白细胞介素-6、肿瘤坏死因子-α、血浆纤溶酶原激活物抑制物-1及尿结缔组织生长因子水平均高于DM组(P<0.05);治疗后DN治疗组上述炎性因子水平及24h尿蛋白定量均较治疗前降低(P<0.05),1,25-二羟维生素D3水平升高(P<0.05),对照组治疗前、后各指标比较差异均无统计学意义(P>0.05)。结论早期DN患者1,25-二羟维生素D3缺乏,炎性因子水平高,补充骨化三醇胶丸可改善患者炎症状态。     

Calcium transport across the colon ascendens and the influence of 1,25-dihydroxyvitamin D3 and dexamethasone

Abstract. Concentration dependence of unidirectional calcium fluxes across the rat colon ascendens were measured in a modified Ussing chamber. Both the mucosa (m) to serosa (s) and the s to m calcium flux exhibited saturation kinetics. The maximum transport rates and the affinity to the transporter of calcium was higher in the m to s direction than that from s to m, resulting in a remarkable net calcium absorption. The results obtained from measurements of unidirectional calcium fluxes in dependence on clamped transepithelial potentials showed that: (i) calcium transport in both direction had a voltage-independent component; (ii) the voltage-independent, i.e. non diffusive fraction of the m to s calcium flux was 3·2 times greater than that in the opposite direction; (iii) the voltage dependent, i.e. diffusional fraction of the m to s calcium flux, was about two times greater than the voltage-dependent fraction of the calcium flux in the s to m direction; and (iv) in the m to s direction 62%, and in the s to m direction 73%, of the total unidirectional flux was voltage-dependent. Dexamethasone, known to enhance sodium and water absorption in the colon, had no significant influence on net calcium absorption but increased the unidirectional calcium fluxes in both directions. The increase in unidirectional calcium fluxes parallel to that of the extracellular marker mannitol suggests that dexamethasone has no influence on the transcellular calcium transport but increases the calcium flux along the paracellular way. Amiloride had no influence on the dexamethasone-induced changes of the epithelial electrical parameters as distinguished from the colon descendens. The increase in calcium and mannitol fluxes after dexamethasone treatment was also unaffected by amiloride. 1,25(OH)₂D₃ had no influence on the kinetics of unidirectional calcium fluxes as distinguished from the colon descendens. The electrical parameters of the tissue and unidirectional mannitol fluxes were also unaffected by 1,25(OH)₂D₃. The mode of calcium transport found in the colon ascendens exhibits characteristics distinct from the transport properties reported for the colon descendens. The heterogeneity of both parts, with respect to the calcium transport, suggests that both colonic segments might have different functions in the final modulation of calcium balance by the intestine.     

慢性肾病患者血1,25-二羟基维生素D3水平变化

目的 研究慢性肾病(CKD)患者血1,25-二羟基维生素D3(维生素D3)水平异常与肾功能受累程度的关系.方法 研究对象包括150例健康对照者和101例CKD患者,检测其血维生素D3、甲状旁腺素、钙、磷、肌酐、清蛋白及尿素氮水平,计算肾小球滤过率(GFR).结果 与对照组相比,第4~5期患者的血维生素D3水平显著降低(P<0.05),第3~5期患者的血甲状旁腺素(PTH)、肌酐及尿素氮(BUN)水平显著升高(P<0.05),第4~5期患者的血磷水平显著升高(P<0.05),各期CKD患者的清蛋白水平均显著降低(P<0.05),仅第5期CKD患者的血钙浓度显著降低(P<0.05).血维生素D3水平与血PTH、尿素氮及肌酐水平呈负线性相关关系,与血钙、清蛋白及GFR水平呈正线性相关关系.结论 第4~5期CKD患者缺乏维生素D3,说明只有当肾功能受损严重时,CKD患者血维生素D3水平才会出现明显异常.此外,本研究还表明CKD患者血维生素D3水平与其他肾功能相关生化指标存在一定的相关性.