鸡蛋黄蛋白质水解产物制备及其对MC3T3-E1细胞增殖分化的影响

鸡蛋黄经脱脂得到蛋黄蛋白质,直接以MC3T3-E1细胞增殖活力为指标,对水解条件进行优化,从而制备蛋黄蛋白质水解产物(egg yolk proteins hydrolysate,EYPH)并对其促MC3T3-E1细胞增殖分化的活性进行研究。结果表明:在对比胰蛋白酶与风味蛋白酶水解过程中,发现单一胰蛋白酶反应得到产物活性最好,胰蛋白酶反应的最佳水解条件为水解时间6 h、水解温度37℃、酶与底物质量比1∶50、p H 8.0。EYPH使MC3T3-E1细胞增殖活力得到提高,为129.89%,细胞周期分析得到S期细胞增加了4.69%,这2个方面证实EYPH对MC3T3-E1细胞的增殖活力有显著的提高。EYPH对MC3T3-E1细胞分化矿化有显著影响,碱性磷酸酶活力提高了9.1%;刺激骨钙素分泌,其含量上升2 mg/mL;形成更多的矿化结节。此外,EYPH还能刺激RUNX2特异性转录因子的基因表达,表达量提高了6.12倍。结果说明EYPH对成骨细胞MC3T3-E1增殖分化有显著的促进作用,因此,EYPH对于抗骨质疏松方面有较好的应用潜力,为开发其功能性食品提供一定的数据参考。     

Effect of selected aromatic medicinal plant sancho (Zanthoxylum schinifolium) on osteoblastic MC3T3-E1 cells

This study was performed to investigate the bioactivity of sancho (Zanthoxylum schinifolium) essential oil (EO) on bone metabolism and its function of osteoblastic MC3T3-E1 cells. The volatile aroma components of sancho EO were collected using a Clevenger-type apparatus by steam distillation extraction method, and determined by GC-MS. β-Phellandrene (22.54%) was the most abundant volatile compound in sancho EO, followed by citronellal (16.48%) and geranyl acetate (11.39%). It increased the collagen and mineralization of osteoblasts (p<0.05), indicating that sancho may help prevent osteoporosis.     

Chemical constituents of Chrysanthemum indicum L. flower oil and effect on osteoblastic MC3T3-E1 cells

The volatile chemical constituents of gamguk (Chrysanthemum indicum L.) produced in Korea, an aromatic medicinal herbaceous plant, were separated by the hydro distillation extraction method, and analyzed by gas chromatography-mass spectrometry. α-Pinene, 1,8-cineol, and chrysanthenone were the predominant aroma components. To investigate the bioactivity of the essential oil from gamguk, which at on bone metabolism, we studied the effects of it on the function of osteoblastic MC3T3-E1 cells were tested. It increased the collagen, alkaline phosphatase activity, and mineralization of osteoblasts significantly (p<0.05), indicating that gamguk may help prevent osteoporosis.     

珍珠贝外套膜胶原蛋白肽及其锌螯合物的体外抑制骨质疏松作用

研究珍珠贝外套膜胶原蛋白肽（pearl oyster mantle collagen peptide,POM-CP）及其锌螯合物（zincchelating pearl oyster mantle collagen peptide,POMCP-Zn）对小鼠前成骨细胞MC3T3-E1增殖分化的影响。结果表 明：噻唑蓝检测结果显示,与空白对照组相比,POMCP-Zn能够显著促进成骨细胞的增殖,而POM-CP对成骨细胞 的增殖性无显著影响;与空白对照组相比,400 μg/mL的POM-CP和10 μg/mL的POMCP-Zn将成骨细胞的碱性磷酸酶 活性分别提高到1.31 倍和1.48 倍;POMCP-Zn还可以提高成骨细胞分化阶段Ⅰ型胶原蛋白、骨钙素蛋白的分泌量, 同时促进成骨细胞矿化阶段骨结节的形成。综上所述,与POM-CP相比,POMCP-Zn对成骨细胞MC3T3-E1的增殖 和分化有较强的促进作用,POMCP-Zn有望成为预防骨质疏松症的潜在功能性食品。     

Effects of a Lactobacillus casei 393 fermented milk product on bone metabolism in ovariectomised rats

The effects of a Lactobacillus casei 393 fermented milk product (FMP) on bone metabolism were examined. FMP (>0.1 mg mL^?1) supplementation significantly increased osteoblastic MC3T3-E1 cells proliferation whereas skim milk powder supplementation did not show any positive effect up to 1 mg mL^?1. The FMP (1%) supplemented ovariectomised rats had increased bone weight, bone mineral density, and bone breaking force compared with control ovariectomised rat. In addition, the activity of tartrate resistant acid phosphatase, a biomarker of osteoclasts, was significantly reduced in the FMP group. Based on the results, the L. casei 393 FMP had a preventative effect on bone loss in ovariectomised rats.     

Activities of MC3T3-E1 cells cultured on gamma-irradiated salmon atelocollagen scaffold

We investigated the effect of gamma-irradiation on the activities of MC3T3-E1 cells cultured on gamma-irradiated salmon atelocollagen (SAC) scaffolds. SAC-cultured samples were irradiated at doses of 10, 15, and 25 kGy. Gamma-irradiation had a significant impact on the activities of MC3T3-E1 cells. The proliferation rates and alkaline phosphatase activities of MC3T3-E1 cells increased with gamma-irradiation dose.     

Effects of soybean ethanol extract on the prostaglandin E2 and interleukin-6 production in osteoblastic cells

We recently found that soybean ethanol extract (0.05 g/l) has a direct stimulatory effect on bone formation in cultured osteoblastic cells in vitro. The present study was conducted to investigate whether soy extract affects the prostaglandin E₂ (PGE₂) and interleukin-6 (IL-6) production of osteoblastic MC3T3-E1 cells. Stimulation with TNF-α resulted in the production of PGE₂ and IL-6 in osteoblasts. Soy extract (0.05 g/l) stimulated the constitutive PGE₂ secretion but had no significant effect on TNF-α-stimulated PGE₂ production. Also, soy extract (0.05 g/l) increased the constitutive IL-6 secretion but had no statistical significance. However, TNF-α-induced IL-6 production was increased significantly (P<0.05). These findings indicate that PGE₂ induction by soy extract in constitutive state may stimulate bone formation whereas IL-6 induction by soy extract in the presence of TNF-α may be important in the inflammatory bone diseases.     

巴戟天多糖的理化性质及其对成骨细胞MC3T3-E1增殖的影响

以巴戟天为原料,通过酶辅助浸提法提取多糖,采用化学和仪器分析法研究多糖的pH、相对黏度、溶解性、光谱特征、空间构像等理化性质,采用噻唑蓝法测定MC3T3-E1细胞增殖率,酶联免疫法测定Ⅰ型胶原蛋白(collagen typeⅠ,COLⅠ)和骨钙素(osteocalcin,OCN)分泌量及碱性磷酸酶(alkaline phosphatase,ALP)活性,评价多糖提取物对成骨细胞增殖的影响。结果表明,巴戟天多糖属于酸性多糖,pH为6.15±0.13,相对黏度为1.26±0.12,具有糖的特性和三股螺旋结构,溶解性较好,不含蛋白质或多肽;巴戟天多糖提取物浓度为50 μg/mL时,MC3T3-E1细胞增殖率极显著增加(P<0.01),达149.67%,COLⅠ和OCN的分泌量极显著增加(P<0.01),分别增加44.98%和85.01%,ALP活性极显著提高(P<0.01),提高了258.95%。表明巴戟天多糖提取物能显著促进MC3T3-E1细胞增殖与分化。     

鲫鱼卵唾液酸糖蛋白化学结构分析及对前成骨细胞MC3T3-E1增殖分化的影响

目的：成熟的鲫鱼卵经提取、分离纯化得到唾液酸糖蛋白（sialyglycoproteins of Carassius auratus eggs,CA-SGP）,分析其化学结构,及对前成骨细胞MC3T3-E1增殖分化的影响。方法：采用水提法从成熟鲫鱼卵中提取水溶性蛋白,经阴离子交换色谱、凝胶过滤色谱分离纯化鲫鱼卵水溶性蛋白得到CA-SGP,测定其基本组成,采用高效凝胶液相色谱和聚丙烯酰胺凝胶电泳测定其分子质量和纯度,PMP柱前衍生高效液相色谱法测定其单糖组成;采用噻唑蓝法测定MC3T3-E1细胞的增殖率,酶联免疫法测定Ⅰ型胶原蛋白（collagen type Ⅰ,COLⅠ）和骨钙素（osteocalcin,OCN）的分泌量及碱性磷酸酶（alkaline phosphatase,ALP）的活性,茜素红染色法测定矿化结节的数量。结果：得到单一的组分CA-SGP,在高效凝胶过滤色谱色谱图上呈现单一的糖和蛋白的检测重叠峰,在凝胶电泳图上呈现单一弥散性条带,其蛋白质含量为14.33%,己糖含量为62.81%,N-乙酰神经氨酸（N-acetylneurainic acid,Neu5Ac）含量为19.72%。单糖组成测定结果显示：CA-SGP主要由甘露糖、葡萄糖胺、半乳糖胺组成,其物质的量比为7.61∶6.70∶1.00;MC3T3-E1细胞与CA-SGP共同孵育后,其增殖率显著增加,COLⅠ和OCN的分泌量分别增加了39.28%和83.06%,ALP活性提高了285.08%,矿化结节的数量增加了96.41%,说明CA-SGP显著促进MC3T3-E1细胞增殖、分化和矿化。结论：CA-SGP为富含Neu5Ac的糖蛋白,具有显著促进MC3T3-E1细胞增殖、分化和矿化的作用,对开发抗骨质疏松功能性食品具有重要意义。     

卵黄高磷蛋白对壳聚糖膜基质上MC3T3-E1细胞增殖的影响

本研究制备了壳聚糖和氧化石墨烯/壳聚糖复合膜,并采用CCK-8法、Annexin V/PI双染法和PI染色检测了蛋黄中提取的卵黄高磷蛋白对MC3T3-E1细胞在该复合膜基质上的生理活动的影响。结果表明:壳聚糖膜对细胞无毒性影响,并通过提高S期细胞所占比例,使MC3T3-E1细胞活性提高了31.92%,抑制细胞凋亡,使凋亡率下降45.78%;氧化石墨烯/壳聚糖膜会提高细胞的凋亡率,氧化石墨烯含量越高,促进凋亡越明显,凋亡率在氧化石墨烯含量为2%时最高,为17.64%;在壳聚糖膜基质上,100 μg/mL卵黄高磷蛋白对MC3T3-E1细胞促进增殖作用最显著(p<0.05),细胞活性提高81.59%,同时降低了细胞的凋亡比例;蛋白浓度为500 μg/mL时,细胞活性下降4.47%,促进了细胞凋亡。综上所述,壳聚糖和低浓度卵黄高磷蛋白均能提高MC3T3-E1细胞的活性。     

金枪鱼鱼骨活性钙对鼠胚胎成骨细胞前体细胞(MC3T3-E1)活力和凋亡作用

目的:以金枪鱼骨为原料,制备柠檬酸-苹果酸钙剂(CMC),研究其对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)的细胞活力、凋亡的影响,并探讨其作用机制。方法:在小鼠胚胎成骨细胞前体细胞(MC3T3-E1)培养液中,分别加入高浓度(1 μg/mL)CMC和低浓度(0.1 μg/mL)CMC,利用MTT法检测对MC3T3-E1细胞活力的影响,流式细胞仪检测血清饥饿诱导的细胞凋亡。结果:高浓度和低浓度的钙对MC3T3-E1细胞活力均有显著的促进作用,24 h的高钙组的细胞增长率(9.67%)优于低钙组(8.95%);36 h和48 h后低钙组的增长率分别为14.96%和20.33%,明显优于36 h和48 h作用后的高钙组(6.23%和1.73%),两组细胞凋亡率与空白组比较差异较小,且均显著低于对照组(p<0.01)。结论:CMC高钙组和CMC低钙组在一定浓度范围内,能促进MC3T3-E1细胞活力显著增强,并且改善血清饥饿诱导的成骨前体细胞凋亡。     

牛骨肽钙螯合物制备、表征及其促MC3T3-E1细胞成骨活性

目的：为制备人体高效吸收利用的补钙剂并探究其对MC3T3-E1细胞成骨活性的影响。方法：对牛骨多肽进行钙螯合处理，以钙螯合能力为考察指标，通过响应面方法确定最佳制备条件；利用红外光谱、X射线衍射和原子吸收等方法探究肽钙螯合物的结构特性及其稳定性；通过细胞实验探究牛骨肽钙螯合物对MC3T3-E1细胞增殖、分化和矿化活性的影响。结果：制备牛骨肽钙螯合物的最佳条件为肽钙质量比2.97∶1、温度62.54℃、pH 9.06。在该条件下，钙螯合能力达到55.65 μg/mg。光谱结构分析结果表明氨基氮、羟基氧和羧基氧是钙主要螯合位点，二者之间主要通过配位键结合，且肽螯合钙后分子质量增加；此外，牛骨肽钙螯合物稳定性受温度影响较小，但对pH值较敏感。MC3T3-E1细胞实验结果表明肽钙螯合物具有明显促MC3T3-E1细胞增殖、分化和矿化活性的作用。结论：制备的牛骨肽钙螯合物具有潜在的促MC3T3-E1细胞成骨活性功效。     

响应面法优化龟甲蛋白提取工艺及其对MC3T3-E1细胞增殖活性

目的:优化龟甲蛋白提取工艺,筛选龟甲促成骨细胞（MC3T3-E1）增殖活性组分。方法:以蛋白含量及对MC3T3-E1细胞增殖率为指标筛选龟甲蛋白提取方法;以料液比、提取温度、时间为自变量,龟甲蛋白含量为因变量,进行单因素提取研究,结合响应面试验设计,获得提取龟甲蛋白最佳工艺;通过超滤技术将龟甲蛋白按分子量分为5个不同组分:总提组分（组分1）,Mw>10 kDa组分（组分2）,Mw:3~10 kDa组分（组分3）,Mw:1~3 kDa组分（组分4）,Mw<1 kDa组分（组分5）,CCK8法测定不同浓度龟甲蛋白（12.5、25、50、100、200 μg/mL）及其不同组分对MC3T3-E1细胞增殖率的影响。结果:磁力搅拌方法得到的龟甲蛋白含量最高,且活性最佳,龟甲蛋白最佳提取条件为料液比1:13（g/mL）、提取温度30℃、提取3 h,此条件下测得龟甲蛋白含量为15.16%;龟甲蛋白不同组分均能促进MC3T3-E1前成骨样细胞增殖,并呈浓度依赖性,在浓度为200 μg/mL时,组分1~5对细胞的增殖率分别为20.58%、25.30%、30.24%、37.89%、38.15%,分子量小于1 kDa的组分促MC3T3-E1细胞增殖活性最佳。结论:本研究为龟甲蛋白的制备工艺提供参考,并证明龟甲蛋白及其不同组分均具有较好的促成骨细胞增殖活性,其中分子量小于1 kDa组分对MC3T3-E1细胞增殖的效果最佳。     

Acteoside inhibits irradiation-mediated decreases in the viability and DNA synthesis of MC3T3-E1 cells

Therapeutic irradiation can cause bone loss, whereas antioxidant supplementation is considered to attenuate irradiation-mediated damages. This study examined whether or not acteoside inhibits irradiation-mediated changes in viability and proliferation of MC3T3-E1 cells. X-ray radiation at >4 Gy not only decreased cell viability and DNA synthesis in the cells, but also increased intracellular levels of reactive oxygen species (ROS) and phosphorylated p66^Shc protein. Irradiation at 8Gy also decreased intracellular levels of reduced glutathione (GSH) and induced G₁ phase arrest of cell cycle progression with the attendant increase of p21 induction. Pretreatment with acteoside inhibited the irradiation-mediated decreases in viability and DNA synthesis by restoring the radiation-mediated changes in the levels of ROS, GSH, p21, and p-p66^Shc to the untreated control levels. These inhibitory activities of acteoside were greater than that of a synthetic antioxidant compound or N-acetyl cysteine did. Collectively, acteoside treatment may prevent irradiation-induced oxidative damages to osteoblasts.     

Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis activities of its metabolites

Imperatorin (IMP) is a major constituent of many herbal medicines and possesses anti-osteoporosis activity. The present research work aimed to study the biotransformation processes of IMP and evaluated the anti-osteoporosis activity of the transformed metabolites. Among 18 strains of filamentous fungi screened, Penicillium janthinellum AS 3.510 exhibited good capability to metabolise IMP to the new derivatives. Ten transformed products were isolated and purified, and their structures were identified accurately based on spectroscopic data. Eight metabolites (2–8 and 10) were novel and previously unreported. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and the lactone ring-opening reaction of furocoumarin skeleton. In addition, anti-osteoporosis activities of all products (1–10) were evaluated using MC3T3-E1 cells. The results showed that products 5 and 8 had the best bioactivities in increasing MC3T3-E1 cell growth. These products could be used in future therapeutic regimens for treating osteoporosis.     

卵黄高磷蛋白磷酸肽及其钙螯合物在双细胞共培养体系中对成骨细胞分化的促进作用

研究了卵黄高磷蛋白磷酸肽（Phosvitin Phosphopeptide,PPP）及其钙螯合物（PPP-Ca）在成骨细胞（MC3T3-E1）和破骨前体细胞（RAW264.7）共培养体系中对成骨细胞分化的调节作用。对细胞毒性、碱性磷酸酶（AKP）、抗酒石酸酸性磷酸酶（TRAP）的活性和TRAP染色进行分析;用RT-PCR技术进一步探究成骨细胞RANKL/OPG通路相关蛋白的mRNA表达情况。结果发现,PPP和PPP-Ca可以使MC3T3-E1体系中的AKP活性分别增加9.5%和12.7%;PPP和PPP-Ca的加入可以使MC3T3-E1体系中OPG基因mRNA的表达量从0.92分别提升至1.25和1.39、使RANKL基因mRNA的表达量从1.00分别提升至1.23和1.45。该研究结果表明,PPP和PPP-Ca可有效促进成骨细胞的分化。实验结果为进一步探索磷酸肽在多细胞模型体系中的作用提供了研究基础,同时为磷酸肽的功能活性开发提供理论依据。     

鲫鱼卵唾液酸糖蛋白制备工艺优化及其促骨形成作用研究

以14种水产动物卵为原料,筛选出具有较高促前成骨MC3T3-E1细胞增殖活性的鲫鱼卵水溶性糖蛋白,采用响应面法优化了鲫鱼卵唾液酸糖蛋白(Sialoglycoprotein of Carassius auratus eggs,Ca-SGP)的提取工艺,并评价Ca-SGP对MC3T3-E1细胞增殖、分化和矿化的影响。结果表明,14种水产动物卵中,鲫鱼卵糖蛋白唾液酸含量最高,且对MC3T3-E1细胞增殖率最高,分别为5.76%和145.71%;采用单因素和响应面试验优化得到制备Ca-SGP的最优提取工艺为:NaCl浓度为0.38 mol/L、提取时间为4.2 h、液料比为1:3.88 w/v,在此条件下,Neu5Ac得率的预测值为592 mg/100 g,验证值为588.47 mg/100 g。Ca-SGP对骨形成作用的研究结果表明,Ca-SGP显著提高MC3T3-E1细胞增殖至135.56%,显著提高COL I、OCN和OPN的分泌量,分别提高到32.23、261.46和85.89 ng/mL,显著提高成骨细胞矿化能力至241.67%,说明Ca-SGP具有显著促骨形成作用(P<0.05)。以上研究为寻找安全、有效抗骨质疏松功能因子提供新途径。     

鲣鱼鱼卵成分分析及其活性糖蛋白提取工艺优化

目的 分析鲣鱼鱼卵营养成分组成,优化鲣鱼鱼卵糖蛋白(Katsuwonus pelamis roe glycoprotein, TGP)制备工艺,并考察其对MC3T3-E1成骨细胞增殖活性的影响。方法 采用食品安全国家标准方法测定鲣鱼鱼卵基本营养成分及其氨基酸、脂肪酸和胆固醇含量;以糖蛋白得率为评价指标,采用单因素实验结合响应面法研究优化TGP制备工艺;应用细胞毒性检测试剂盒(cell counting kit-8, CCK-8)检测TGP对MC3T3-E1细胞增殖活性的影响。结果 鲣鱼鱼卵中水分含量(72.79±0.33) g/100 g,蛋白质含量(21.09±0.03) g/100 g,脂肪含量(3.91±0.06)g/100g,灰分含量(1.50±0.01)g/100g,总糖含量(0.50±0.01)g/100g。鱼卵中氨基酸总量(727.38±8.77) mg/g,其中必需氨基酸占39.42%,鲜味氨基酸占30.68%,且谷氨酸含量最高(84.61±0.98) mg/g。鱼卵中含有17种脂肪酸,其中二十二碳六烯酸(docosahexaenoi cacid,DHA)和二十碳五烯酸...     

Sialoglycoprotein isolated from Carassius auratus eggs promotes osteoblast differentiation via targeting the p38 mitogen‐activated protein kinase‐dependent Wnt/β‐catenin and BMP2/Smads pathways

In the current study, sialoglycoprotein isolated from eggs of Carassius auratus promoted MC3T3‐E1 cells proliferation and differentiation, as assessed by MTT, mineralized nodule formation in vitro, as well as new osteoid formation in neonatal mouse calvarias ex vivo. Further research revealed that Ca‐SGP facilitated osteogenesis via activating BMP2/Smads, Wnt/β‐catenin, and p38MAPK signaling pathways. What is more, p38MAPK inhibitor SB203580 down‐regulated related mRNA and protein expression of BMP2/Smads and Wnt/β‐catenin signaling pathways, suggesting that p38MAPK pathway might be important for activation of BMP2/Smads and Wnt/β‐catenin pathways in Ca‐SGP‐induced osteoblastic differentiation. In conclusion, it was demonstrated that Ca‐SGP promotes osteoblasts differentiation via activating p38MAPK‐dependent BMP2/Smads and Wnt/β‐catenin signaling pathways, which may provide basis for the use of Ca‐SGP as a potential agent to treat bone loss‐associated diseases such as osteoporosis.

Practical application

Carassius auratus eggs are one of the major by‐products during fish processing and contain sialoglycoprotein which plays an important role in biological functions. However, they have not been high‐value utilized. This study demonstrated that Sialoglycoprotein isolated from eggs of C. auratus (Ca‐SGP) promoted MC3T3‐E1 cells proliferation, differentiation and mineralized nodule formation in vitro, as well as neonatal mouse calvarias formation ex vivo, which may provide basis for a novel application of C. auratus eggs as a functional food used to accelerate bone formation.     

桑白皮提取物对破骨细胞 和成骨细胞活性的影响

目的:探究桑白皮提取物对破骨细胞和成骨细胞活性的影响。方法:通过回流提取和大孔树脂、聚酰胺柱层析制备桑白皮的四种提取物(水提物W、醇提物E、纯化提取物R和PA),采用可见分光光度法对不同提取物中的黄酮含量进行测定。采用1α,25-(OH)₂VitD₃诱导兔原代骨髓细胞以获得破骨细胞,诱导培养8 d后进行TRAP染色,通过TRAP+细胞计数考察桑白皮提取物对破骨细胞形成的影响。培养MC3T3-E1 Subclone 14细胞,采用MTT法考察桑白皮提取物对成骨细胞增殖的影响。结果:桑白皮四种提取物中黄酮的含量为:PA(37.23%) > R(26.80%) > E(21.83%) > W(15.38%)。提取物PA(10、20、50 mg/L)、R(20、50 mg/L)、E(10、20 mg/L)和W(20 mg/L)实验组与空白对照组相比,TRAP+细胞个数减少,且具有显著性差异(P<0.01或P<0.05)。MTT法增殖试验结果表明,四种提取物各剂量组均能促进MC3T3-E1 Subclone 14的增殖,与空白组相比具有极显著性差异(P<0.01),且促增殖作用随黄酮含量增加而增强。结论:桑白皮提取物能够抑制破骨细胞的形成,且对成骨细胞的增殖有促进作用,黄酮类化合物可能是桑白皮发挥活性作用的重要成分。