Production of polymeric micelles by microfluidic technology for combined drug delivery: Application to osteogenic differentiation of human periodontal ligament mesenchymal stem cells (hPDLSCs)

The current paper reports the production of polymeric micelles (PMs), based on pluronic block-copolymers, as drug carriers, precisely controlling the cellular delivery of drugs with various physico-chemical characteristics. PMs were produced with a microfluidic platform to exploit further control on the size characteristic of the PMs.     

微小 RNA-218-5p靶向 Jagged 1调控人牙周膜干细胞的活性和骨向分化

目的 研究微小RNA-218-5p(miR-218-5p)对人牙周膜干细胞(hPDLSCs)活性和骨向分化的影响并探讨其机制,为牙周炎的治疗提供研究基础.方法 将anti-miR-218-5p组(转染anti-miR-218-5p)、anti-miR-NC组(转染anti-miR-NC)、pcDNA组(转染pcDNA)、pcDNA-JAG1组(转染pcDNA-JAG1)、anti-miR-218-5p+si-NC组(共转染anti-miR-218-5p和si-NC)、anti-miR-218-5p+si-JAG1组(共转染anti-miR-218-5p和si-JAG1),用脂质体法转染至hPDLSCs细胞;运用实时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测细胞中miR-218-5p、Ⅰ型胶原蛋白(Col-1)、骨钙素、Runt相关转录因子2(Runx2)的表达;蛋白质印迹法(Westrn blotting)检测细胞中Jagged 1(JAG1)的蛋白表达;MTT法检测细胞活性;茜素红染色实验检测细胞的矿化结节;双荧光素酶报告基因检测实验检测细胞的荧光活性.结果 与anti-miR-NC组相比,anti-miR-218-5p组hPDLSCs细胞培养48、72 h时,细胞活性升高[48 h:(0.44±0.04)比(0.62±0.06);72 h:(0.53±0.05)比(0.83±0.08);P<0.001],细胞的矿化结节明显升高,Col-1[(0.26±0.03)比(0.74±0.07)]、骨钙素[(0.21±0.02)比(0.54±0.05)]、Runx-2[(0.29±0.03)比(0.61±0.06)]蛋白相对表达量均显著升高(均P<0.001).与pcDNA组相比,pcDNA-JAG1组hPDLSCs细胞培养48、72 h时,细胞活性显著升高[48 h:(0.42±0.04)比(0.59±0.05);72 h:(0.55±0.05)比(0.81±0.08);P<0.001],Col-1[(0.34±0.03)比(0.71±0.07)]、骨钙素[(0.23±0.02)比(0.48±0.04)]、Runx-2[(0.25±0.03)比(0.56±0.05)]蛋白表达量均显著升高(均P<0.001).miR-218-5p可靶向调控JAG1的表达.抑制JAG1可逆转抑制miR-218-5p对hPDLSCs的活性和骨向分化作用.结论 抑制miR-218-5p可促进人牙周膜干细胞活性和骨向分化,其机制与靶向JAG1有关.     

1,25(OH)2D3在牙乳头干细胞成骨分化期的作用研究

摘要 目的：研究1,25(OH)2D3在牙乳头干细胞向成骨细胞分化过程中的作用角色。方法：分离培养牙乳头干细胞,培养基中添加不同浓度1,25(OH)2D3,MTT法检测细胞生长速度,流式细胞术检测细胞增殖周期变化,western blot方法检测核因子-k B受体活化剂配体（RANKL）、骨保护素（OPG）和维生素D受体（VDR）的蛋白质表达水平;siRNA技术沉默VDR表达后,检测其对RANKL和OPG蛋白质表达的影响。结果：MTT和流式细胞术检测结果显示1,25(OH)2D3对牙乳头干细胞的增殖没有明显作用;western blot结果显示RANKL、OPG和VDR的蛋白质表达与1,25(OH)2D3浓度具有剂量相关性;siRNA沉默VDR表达后,RANKL和OPG的蛋白质表达水平均有不同程度下降。结论：1,25(OH)2D3通过调节VDR水平影响牙乳头干细胞向成骨细胞方向的分化过程。     

1,25-二羟维生素D_3对人胃腺癌细胞诱导分化的实验研究

目的观察1,25-二羟维生素D3对胃腺癌MGC-803细胞的诱导分化作用。方法1,25-二羟维生素D3(10-6、10-5、10-4mmol.L-1)作用于MGC-803细胞后,应用四甲基偶氮唑蓝(MTT)法测定细胞增殖能力、平板克隆试验测定细胞集落形成率、流式细胞仪测定细胞周期、TRAP-银染法测定端粒酶活性。结果1,25-二羟维生素D3作用24、48、72和96h后胃腺癌细胞生长均明显受抑制,48h后细胞周期出现向G0/G1期移行的特征性动力学改变,端粒酶活性明显受到抑制。细胞集落形成率明显下降(P<0.05)。结论1,25-二羟维生素D3对人胃腺癌细胞有诱导分化作用。     

CTGF在单侧输尿管梗阻大鼠肾组织中的表达以及活性维生素D_3干预治疗的研究

目的：观察单侧输尿管梗阻大鼠肾脏组织中CTGF的表达以及活性维生素D3（1,25-（OH）2D3）对其表达的影响。探讨1,25-（OH）2D3对大鼠肾间质纤维化的调节作用。方法：随机采用体重180~220g的清洁级Wistar大鼠,雄性,36只,随机分为假手术组（n=12）、UUO模型组（n=12）、活性维生素D3组（n=12）。造膜后活性维生素D3组给予罗盖全（骨化三醇）灌胃（3ng/100g）,假手术组及UUO模型组给予等量生理盐水灌胃。给药后第14天,28天分批（n=6）处死,取造膜侧肾脏。用HE染色及Masson染色方法观察肾脏组织纤维化程度,用免疫组化法测定结缔组织生长因子（CTGF）在各组大鼠造膜侧肾脏的表达情况。结果：与假手术组比较,UUO模型组大鼠的肾脏组织中CTGF的表达有显著增高（P〈0.05）,而在活性维生素D3治疗组中,CTGF的表达低于UUO模型组（P〈0.05）,肾间质纤维化程度也明显小于模型组（P〈0.01）。结论：CTGF在UUO大鼠肾脏中表达明显增高,1,25（OH）2D3能有效抑制UUO大鼠肾脏组织CTGF的表达,从而减轻UUO大鼠肾脏组织纤维化程度。     

1,25（OH）2D3对人肾小球系膜细胞增殖及PCNA表达的影响

目的探讨1,25-二羟基维生素D3[1,25（OH）₂D₃]对人肾小球系膜细胞中增殖细胞核抗原（PCNA）表达及细胞增殖的影响。方法体外培养人肾小球系膜细胞,取传代培养至3⁸代细胞随机分为4组:正常对照组（加含5%胎牛血清DMEM培养基）,增殖对照组（EGF组,加10μg/L的EGF）,一般干预组[VD组,加10^-8mol/L 1,25（OH）₂D₃],增殖干预组[EGF+VD组,加10μg/L EGF及10^-8mol/L1,25（OH）₂D₃],均作用48 h。流式细胞术检测各组细胞周期;Western blot检测各组PCNA表达的情况。结果 （1）细胞周期。与正常对照组相比,EGF组G₁期细胞明显减少,S、G2/M期细胞增多,增殖指数（PI）较高;VD组G₁期细胞明显增多,S、G₂/M期细胞减少,PI较低;与EGF组相比,VD组和EGF+VD组G₁期细胞增多,S、G₂/M期细胞减少,PI较低,差异均有统计学意义。（2）系膜细胞中PCNA蛋白的表达。与正常对照组相比,EGF组PCNA的表达较高,VD组PCNA的表达较低;与EGF组相比,VD组和EGF+VD组PCNA的表达较低。结论 1,25（OH）₂D₃通过阻滞细胞周期、抑制PCNA的表达,从而抑制人肾小球系膜细胞的增殖。     

1,25-二羟维生素D3对子宫内膜癌HEC-1-A细胞增殖与S期激酶相关蛋白及抑癌基因p27表达的影响

Objective To investigate the expressions of vitamin D receptor (VDR) in endometrial carcinoma cell HEC-1-A;to study the effect of 1,25-(OH)2D3 on prolifetation of human endometrial carcinoma cell HEC-1-A and the mechanism of detecting the expression of S-phase kinase associated protein 2 ( Skp2 ) and p27 gene.Methods Expressions of VDR in human endometrial carcinoma cell HEC-1-A were detected by immunohistochemical method. HEC-1-A human endometrial carcinoma cell line were cultured in vitro and treated with different concentrations of 1,25-( OH)2D3 for 1-5 days. Cell proliferation was evaluated by CCK-8 method, distribution of cell cycle were determined by flow cytometry. The expression level of Skp2/p27 mRNA was examined by real-time quantitative polymerase chain reaction (RT-qPCR). The expression level of Skp2/p27 protein was finally detected by Western blotting. Results (10-8-10-6) mol/L 1,25-(OH)2D3 inhibited the proliferation of HEC-1-A cell line,and inhibitory rate was positively associated with concentration of 1,25-( OH)2D3 and treatment time. 1,25-(OH)2D3 increased the rates of G0/G1, decreased the rates of S and G2/M and also decreased cells PI (P ＜0.01 ). The mRNA and protein expression of Skp2 in HEC-1-A cell lines after 1,25 (OH)2 D3 treatment decreased.The level of p27 mRNA was decreased but the level of p27 protein was significantly increased. Conclusions 1 ,25-(OH)2D3 can inhibit the growth of HEC-1-A cell line. It may inhibit cancer cell proliferation through down-regulation of Skp2 gene and increase p27 stability by decreasing the abundance of Skp2.     

1,25(OH)2D3对肺炎链球菌感染的肺泡上皮细胞功能的影响

目的 观察1,25-双羟维生素D3(1,25(OH)2D3)通过miR-124-3p调控磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/AKT)通路对肺炎链球菌(SP)感染的肺泡上皮细胞功能的影响,探讨其作用机制.方法 体外培养肺泡上皮细胞A549,肺炎链球菌刺激后,随机分为10,20,40,80,160 ng·m...     

1,25-二羟维生素D3对子宫内膜癌HEC-1-A细胞增殖与S期激酶相关蛋白及抑癌基因p27表达的影响

Objective To investigate the expressions of vitamin D receptor (VDR) in endometrial carcinoma cell HEC-1-A;to study the effect of 1,25-(OH)2D3 on prolifetation of human endometrial carcinoma cell HEC-1-A and the mechanism of detecting the expression of S-phase kinase associated protein 2 ( Skp2 ) and p27 gene.Methods Expressions of VDR in human endometrial carcinoma cell HEC-1-A were detected by immunohistochemical method. HEC-1-A human endometrial carcinoma cell line were cultured in vitro and treated with different concentrations of 1,25-( OH)2D3 for 1-5 days. Cell proliferation was evaluated by CCK-8 method, distribution of cell cycle were determined by flow cytometry. The expression level of Skp2/p27 mRNA was examined by real-time quantitative polymerase chain reaction (RT-qPCR). The expression level of Skp2/p27 protein was finally detected by Western blotting. Results (10-8-10-6) mol/L 1,25-(OH)2D3 inhibited the proliferation of HEC-1-A cell line,and inhibitory rate was positively associated with concentration of 1,25-( OH)2D3 and treatment time. 1,25-(OH)2D3 increased the rates of G0/G1, decreased the rates of S and G2/M and also decreased cells PI (P ＜0.01 ). The mRNA and protein expression of Skp2 in HEC-1-A cell lines after 1,25 (OH)2 D3 treatment decreased.The level of p27 mRNA was decreased but the level of p27 protein was significantly increased. Conclusions 1 ,25-(OH)2D3 can inhibit the growth of HEC-1-A cell line. It may inhibit cancer cell proliferation through down-regulation of Skp2 gene and increase p27 stability by decreasing the abundance of Skp2.     

盐酸二甲双胍对成骨细胞增殖、BMP-2及Cbfa-1基因表达的影响

目的初步研究二甲双胍（MF）在体外对小鼠颅盖骨成骨细胞增殖、骨形态发生蛋白一2（BMP一2）及核心结合因子（Cbfa一1）mRNA表达的影响,探讨二甲双胍对骨代谢的可能作用机制。方法（1）分离培养原代颅盖骨成骨细胞并对其进行鉴定。（2）以乳鼠成骨细胞为体外实验模型,不同浓度（0、50、100、200、400Ixmol／L）的MF干预体外培养的成骨细胞24h后,M1vr法检测成骨细胞的增殖能力,实时荧光定量PCR法检测成骨细胞BMP-2及Cbfa-1基因表达。结果二甲双胍干预成骨细胞24h后,可促进成骨细胞的增殖,在浓度400μxmol／L的OD值最大为0．298±0．047（P〈0．05）;可促进BMP-2及Cbfa-1mRNA的表达,呈剂量效应关系。结论二甲双胍可促进成骨细胞的增殖和分化,可能通过调节BMP-2及Cbfa-1的表达,从而促进骨的形成。     

1,25-二羟维生素D3对子宫内膜癌HEC-1-A细胞增殖与S期激酶相关蛋白及抑癌基因p27表达的影响

目的 观察1,25-二羟维生素D3对人子宫内膜癌HEC-1-A细胞增殖及S期激酶相关蛋白（Skp2）基因、抑癌基因p27表达的影响,探讨其抗癌机制.方法 应用免疫组织化学方法检测人子宫内膜癌HEC-1-A细胞表达维生素D受体（VDR） 选用人子宫内膜癌HEC-1-A细胞进行体外培养,用CCK-8法测定不同浓度1,25-二羟维生素D3作用细胞后不同时间后细胞生长抑制率,流式细胞仪进行细胞周期分析,采用荧光实时定量聚合酶链反应（PCR）及蛋白质印迹法检测Skp2/p27基因表达的变化.结果 人子宫内膜癌HEC-1-A细胞表达VDR,且定位于细胞核内 1×（10-8～10-6）mol/L浓度的1,25-二羟维生素D3作用于HEC-1-A细胞1～5 d范围内各不同浓度处理组细胞生长增殖均受到明显抑制,并且随着作用浓度增加,细胞增殖能力逐渐下降 1,25-二羟维生素D3能增加G0/G1期细胞比例,降低S、G2/M期细胞比例,从而降低细胞增殖指数（P＜0.01） 在mRNA、蛋白质水平上,能降低Skp2表达,而p27mRNA减少,p27蛋白表达明显增加.结论 1,25-二羟维生素D3对人子宫内膜癌HEC-1-A细胞有抑制作用,可能是通过转录水平下调Skp2基因表达,同时由于Skp2的减少增加p27蛋白稳定性,减少其降解,从而产生抑癌作用.     

1,25二羟维生素D_3对卵巢癌细胞株HO8910增殖及Skp_2/p27蛋白表达的影响

目的:观察1,25二羟维生素D3[1,25(OH)2D3]对人卵巢癌细胞株HO8910增殖的影响,并且检测其作用下S期激酶相关蛋白(Skp2)、抑癌基因p27的蛋白表达情况,以探讨其作用机制。方法:选用人卵巢癌细胞株HO8910进行体外培养,用MTT比色法测定不同浓度D3[1,25(OH)2D3]对细胞株作用不同时间后细胞生长抑制率,流式细胞仪进行细胞周期分析,免疫细胞化学染色检测Skp2及p27蛋白表达情况。结果:1,25(OH)2D3在160×10-9mol/L以上时,对细胞有抑制作用(P<0.05),且呈浓度时间依赖性;1,25(OH)2D3能增加G0/G1期细胞比例,降低S、G2/M期细胞比例,从而降低细胞增殖指数(P<0.01);能降低Skp2表达,增加p27蛋白表达(P<0.01)。结论:1,25(OH)2D3对人卵巢癌细胞株HO8910有抑制作用,可能通过下调Skp2表达、上调p27蛋白表达,从而抑制癌细胞增殖。     

Preventive effects of 1,25-（OH）2VD3 against ConAinduced mouse hepatitis through promoting vitamin D receptor gene expression

Aim:

To investigate the immunosuppressive effects of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂VD₃) on concanavalin A (ConA)-induced hepatitis and elucidate the action mechanism.

Methods:

Female BALB/C mice were intravenously administered ConA (20 mg/kg) to induce acute immunological liver injury. Liver damage was evaluated in respect to serum alanine transaminase (ALT) level and liver histological changes. The proliferation of splenocytes was measured by using [³H]-thymidine incorporation. The cytokine level in the cultured splenocyte supernatant was determined by using enzyme-linked immunosorbent assays (ELISAs). The percentage of different splenic T cell subtypes was analyzed by using flow cytometry. The expression of splenic vitamin D receptor (VDR) mRNA and protein was detected by using real-time qRT-PCR and Western blot, respectively.

Results:

1,25-(OH)₂VD₃ (2.5 μg/kg, ip) significantly decreased the serum ALT levels and markedly attenuated the histological liver damage. The beneficial effect of 1,25-(OH)₂VD₃ was associated with: (i) inhibition of CD4⁺ T cell activation; (ii) reduction of interferon-γ (IFN-γ) and elevation of both IL-4 and IL-5 in supernatants of cultured splenocytes; and (iii) elimination of activated T cells by increasing VDR mRNA and protein expression in the spleen.

Conclusion:

1,25-(OH)₂VD₃ had a significant protective effect against ConA-induced hepatitis, and its mechanism of action was associated with down-regulation of T cell-mediated immunity and up-regulation of VDR gene expression.     

1,25-二羟维生素D_3对哮喘大鼠BALF中Eotaxin及IL-8表达的影响

目的观察1,25-(OH)2D3对哮喘大鼠BALF中Eotaxin及IL-8表达的影响。方法 50只Wistar大鼠随机分为正常对照组(N)、哮喘组(A)、1,25-(OH)2 D3组(VD)、布地奈德组(B)及1,25-(OH)2 D3+布地奈德联合治疗组(L),每组10只。用ELISA法检测BALF中Eotaxin及IL-8的表达水平。结果各组Eotaxin和IL-8表达比较:A组较其他各组明显增高(P<0.01、P<0.05);VD组较A组明显降低(P<0.05),但仍高于N组、B组及L组(P<0.01);L组较A组、VD组及B组明显减低(P<0.05、P<0.01)。结论哮喘急性发作时,BALF中Eotaxin及IL-8的表达水平显著增高,1,25-(OH)2D3有效抑制了哮喘时的Eotaxin和IL-8的表达水平,与布地奈德联合治疗作用更为明显。     

Brain uptake and CNS levels of 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)urea (HECNU)

The uptake of ¹⁴C-labeled 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl) urea (HECNU) into the brain was investigated in the rat after intracarotid injection according to the method of OLDENDORF, as well as in cisternal cerebrospinal fluid obtained by suboccipital puncture after i.v. injection of the drug.The brain uptake index was 31.9 ± 2.9%. Cerebrospinal fluid/blood quotients after i.v. injection were 0.82 at 10 min and 1.10 at 60 min. The results of both methods clearly show that HECNU, in spite of its hydrophilic property, easily penetrates the blood-brain barrier.     

槲皮素-3-O-芹菜糖基芦丁糖苷对新生大鼠海马神经前体细胞增殖的影响

目的探讨一种新型抗抑郁剂槲皮素-3-O-芹菜糖基芦丁糖苷(代号:CTN-986)对神经前体细胞增殖的影响。方法分离培养新生大鼠海马神经前体细胞,运用免疫细胞化学的方法对所培养的细胞特性进行鉴定。用MTT法和3H-胸腺嘧啶核苷参入法分别观察CTN-986对新生大鼠海马神经前体细胞增殖的影响,并初步探讨其作用机制。结果所培养的细胞具有神经前体细胞的特性,药物研究发现,氟西汀和CTN-986(2~250μmol.L-1)作用4 d,可以促进神经前体细胞的增殖;同时给予5-HT1A受体特异性拮抗剂WAY-100635(0.1μmol.L-1)可以对抗CTN-986诱导的促神经前体细胞的增殖。结论CTN-986可以促进海马神经前体细胞的增殖,并且该作用的发挥与激活5-HT1A受体关系密切。     

Comparative effects of 1α‐hydroxyvitamin D3 and 1,25‐dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(‐/‐) mice

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P‐gp expression. The present study compared the equimolar efficacies of 1α‐hydroxyvitamin D₃ [1α(OH)D₃] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half‐life that is converted to 1,25(OH)₂D₃, and 1,25(OH)₂D₃ on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)‐directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(‐/‐)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D₃ and 1,25(OH)₂D₃, 1α(OH)D₃ treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(‐/‐) mice compared to 1,25(OH)₂D₃ treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P‐gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D₃ treatment. The higher activity of 1α(OH)D₃ was explained by its rapid conversion to 1,25(OH)₂D₃ in tissue sites, furnishing higher plasma and tissue 1,25(OH)₂D₃ levels compared to following 1,25(OH)₂D₃‐treatment. In conclusion, 1α(OH)D₃ exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)₂D₃ in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

2β(3羟丙氧基)骨化三醇对人肝癌细胞周期阻滞及P~(27kip1)蛋白的诱导表达

目的探讨2β(3羟丙氧基)骨化三醇(ED-71)诱导人肝癌细胞HepG2生长抑制和细胞周期G1阻滞,及对抑癌基因P27kip1表达的影响。方法培养HepG2,应用四甲基偶氮唑盐(MTT)比色法观察ED-71对HepG2的生长抑制作用,流式细胞术分析细胞周期,以Western-blot检测HepG2细胞中P27kip1蛋白的表达水平。结果ED-71处理后HepG2的生长缓慢,细胞生长受到明显抑制。细胞阻滞于G1期(80.6±2.6,48.7±3.0,P<0.05),P27kip1蛋白表达水平增强(0.11±0.06,0.67±0.08,P<0.05)。结论ED-71抑制人肝癌细胞株HepG2的生长,诱导人肝癌细胞分化,使细胞阻滞于G1期,可能与ED-71诱导人肝癌细胞中抑癌基因P27kip1蛋白的表达有关。     

1-(5-异喹啉磺酰基)-2-甲基哌嗪(H-7)对肺纤维化大鼠肺单核细胞趋化蛋白-1 mRNA表达的影响

研究蛋白激酶C(PKC)抑制剂 1 (5 异喹啉磺酰基 ) 2 甲基哌嗪 (H 7)对肺纤维化大鼠单核细胞趋化蛋白 1(MCP 1)mRNA表达的影响 ,为临床治疗肺纤维化提供理论基础 .博来霉素致大鼠肺纤维化动物模型 ,腹腔注射H 7后 ,定量RT PCR方法检测其对肺组织MCP 1mRNA表达的影响 ;测定实验组和对照组肺组织匀浆内羟脯氨酸的含量 ,观察肺组织病理学变化并计数单核细胞 .结果发现 ,H 7能显著抑制大鼠肺纤维化组织MCP 1mRNA表达和单核细胞的聚集以及羟脯氨酸的含量 .结果表明 ,H 7通过抑制肺组织MCP 1mRNA的表达 ,对博来霉素致肺纤维化有明显的防治作用 .