Optimal concentration of mesenchymal stem cells for fracture healing in a rat model with long bone fracture

BACKGROUND There is still no consensus on which concentration of mesenchymal stem cells(MSCs)to use for promoting fracture healing in a rat model of long bone fracture.AIM To assess the optimal concentration of MSCs for promoting fracture healing in a rat model.METHODS Wistar rats were divided into four groups according to MSC concentrations:Normal saline(C),2.5×10^(6)(L),5.0×10^(6)(M),and 10.0×10^(6)(H)groups.The MSCs were injected directly into the fracture site.The rats were sacrificed at 2 and 6 wk post-fracture.New bone formation[bone volume(BV)and percentage BV(PBV)]was evaluated using micro-computed tomography(CT).Histological analysis was performed to evaluate fracture healing score.The protein expression of factors related to MSC migration[stromal cell-derived factor 1(SDF-1),transforming growth factor-beta 1(TGF-β1)]and angiogenesis[vascular endothelial growth factor(VEGF)]was evaluated using western blot analysis.The expression of cytokines associated with osteogenesis[bone morphogenetic protein-2(BMP-2),TGF-β1 and VEGF]was evaluated using real-time polymerase chain reaction.RESULTS Micro-CT showed that BV and PBV was significantly increased in groups M and H compared to that in group C at 6 wk post-fracture(P=0.040,P=0.009;P=0.004,P=0.001,respectively).Significantly more cartilaginous tissue and immature bone were formed in groups M and H than in group C at 2 and 6 wk post-fracture(P=0.018,P=0.010;P=0.032,P=0.050,respectively).At 2 wk post fracture,SDF-1,TGF-β1 and VEGF expression were significantly higher in groups M and H than in group L(P=0.031,P=0.014;P<0.001,P<0.001;P=0.025,P<0.001,respectively).BMP-2 and VEGF expression were significantly higher in groups M and H than in group C at 6 wk postfracture(P=0.037,P=0.038;P=0.021,P=0.010).Compared to group L,TGF-β1 expression was significantly higher in groups H(P=0.016).There were no significant differences in expression levels of chemokines related to MSC migration,angiogenesis and cytokines associated with osteogenesis between M and H groups at 2 and 6 wk post-fracture.CONCLUSION The administration of at least 5.0×10^(6)MSCs was optimal to promote fracture healing in a rat model of long bone fractures.

Effect of thyroid hormone on serum NO concentration and iNOS activity of intestinal mucosa in septic rats

Objective To investigate the effect of exogenous thyroid hormone on serum NO and iNOS activity of intestinal mucosa in septic rats. Methods Septic model was established by cecal ligation puncture (CLP) in male SD rats. Triiodothyronine ( T3 ) was administered intraperitoneally to correct the low T3 syndrome of septic rats. Blood was collected to examine serum NO and thyroid hormone concentration. Intestinal mucosa iNOS activity was assayed using immunochemical stain. Results Mortality rate in the prevention group was significantly lower than the septic group (Log rank = 3. 85, P 【 0.05). Serum NO concentration was significantly lower in the prevention group (F=19.6,F【0.01). The degree of inflammatory injury of intestinal mucosa was much milder in the prevention group than in the septic group (x2 = 5.303,P【0. 05). Mucosa iNOS activity was also significantly lower in the prevention group (x2 = 4. 876, P【0. 01). Conclusion Thyroid hormone protects the intestinal mucosa barrier inhibiting the expression of

Chronic Toxicity Study in Rats Orally Exposed to Mulberry Sea-Buckthorn Beverage Concentrate

Objective: This study is designed to observe the chronic toxicity after the administration of mulberry sea-buckthorn beverage concentrate for 3 months on rats and to predict the possible adverse effect and the potential toxicity target organs. Method: The rats (SPF level) were randomly divided into high-dose (20 mL/kg BW), middle-dose (10 mL/kg BW), low-dose (5 mL/kg BW) groups and negative control group (20 mL/kg BW of purified water) with 30 rats in each group. Each group was orally given mulberry sea-buckthorn beverage concentrate for 3 months and recovered by stop feeding samples for 2 weeks for a recovery observation. The rats’ general condition, the organ coefficient, the indexes of hematology and blood biochemistry and the histological changes of the main organs were determined. Result: The appearance and behavior of activity in rats showed no anomalies in all these groups and all the rats put on weight during this period. Comparing to the negative control group, no obvious differences were observed in the weekly weight and organ coefficient of each dose group. After 3 months of administration, HGB in both mulberry sea-buckthorn beverage concentrate low-dose group and high-dose group were increased. No significant differences were observed in the indexes of hematology after 2 weeks of recovery. CREA in low-dose, middle-dose and high-dose groups were significantly increased after 3 months of administration and it remained in the high level in middle-dose and high-dose group even after 2 weeks of recovery. No drug-related lesions were observed in the histological changes of major organs. Conclusion: The results show that long term use of mulberry concentrated sea-buckthorn beverage can lead to increased CREA, which suggested kidney toxicity. Although no obvious pathological change was found in kidney, we should pay attention to chronic kidney damage in the further research.

三七总皂苷上调浓缩生长因子释放促进大鼠骨折愈合

背景:研究表明,三七总皂苷和浓缩生长因子均能促进骨折愈合,但是目前关于两者共同干预骨折愈合的研究较少,三七总皂苷可能通过在某段时间内促进浓缩生长因子相关因子的释放来达到加快骨折愈合的目的。目的:观察三七总皂苷对浓缩生长因子释放,以及对大鼠骨折愈合的影响。方法:8周龄SD大鼠18只,编号后随机分为三七总皂苷组、模型对照组以及空白组。三七总皂苷组给予三七总皂苷灌胃2周,模型对照组给予生理盐水2 mL灌胃2周,空白组正常饲喂。2周后三七总皂苷组与模型对照组同时采血离心获取浓缩生长因子备用,继续正常饲喂1周,然后对所有大鼠进行股骨骨折造模处理,三七总皂苷组与模型对照组植入自体浓缩生长因子,并用ELISA法检测1 h及1,3,5,7,9,11 d生长因子释放量;通过观察术后2个月X射线片及骨组织苏木精-伊红染色结果,评估骨折愈合情况。结果与结论:①生长因子释放情况:三七总皂苷组的血管内皮生长因子A和转化生长因子β在第7,9,11天的释放浓度高于模型对照组(P<0.05),血小板衍生生长因子BB在第5,9,11天的释放浓度高于模型对照组(P<0.05),碱性成纤维细胞生长因子在1-11天的释放浓度均高于模型对照组(P<0.05);②骨折愈合情况:术后2个月行X射线片检查,三七总皂苷组的骨折愈合情况优于模型对照组,两组均优于空白组;取材苏木精-伊红染色发现,三七总皂苷组成骨细胞生成量大于模型对照组,两组均大于空白组;③提示三七总皂苷可以上调浓缩生长因子相关因子的释放浓度,三七总皂苷干预后的浓缩生长因子对促进大鼠骨折愈合更有优势。

超高效液相色谱串联质谱法考察大鼠体内索凡替尼药代动力学

目的建立测定大鼠血浆中索凡替尼浓度的超高效液相色谱串联质谱(UPLC-MS/MS)法。方法血浆样品经沉淀蛋白处理后进样,以卡马西平为内标。色谱柱为Waters Acquity UPLC®BEH C_(18)柱(50 mm×2.1 mm,1.7µm),流动相为甲酸-氨水-水(1∶1∶1000,V/V/V)-乙腈(梯度洗脱),流速为0.4 mL/min,柱温为40℃,进样量为1µL,离子源为电喷雾电离(ESI),在正离子、多反应监测模式下检测,气帘气压为35 psi,雾化气压为30 psi,喷雾电压为5500 V,离子对质荷比(m/z)分别为m/z 481.2→329.0(索凡替尼)和m/z 237.2→194.0(卡马西平)。结果索凡替尼及内标卡马西平的保留时间分别为0.93 min和0.94 min。索凡替尼的质量浓度在0.1~500 ng/mL范围内与索凡替尼和卡马西平峰面积的比值线性关系良好(r=0.9972,n=9);精密度、稳定性、准确度、基质效应试验的结果均符合要求;定量下限为0.1 ng/mL。按体质量2 mg/kg单次灌胃给药,达峰时间(t_(max))约为1.33 h,峰浓度(C_(max))约为5.92 ng/mL,半衰期(t_(1/2))约为5.31 h,平均滞留时间(MRT)约为4.02 h;给药4 h后,索凡替尼的肝脏浓度(201.2 ng/mL)远高于血浆浓度(3.57 ng/mL)。结论该方法简便、快速、准确、灵敏,可用于大鼠血浆中索凡替尼浓度的快速测定。索凡替尼易在肝脏中蓄积。

山奈酚对缺氧/复氧损伤诱导心肌细胞凋亡的影响及机制研究

目的:探究山奈酚对缺氧/复氧损伤诱导心肌细胞凋亡的影响和机制。方法:利用随机数字法将H9C2大鼠心肌细胞随机分为三组,对照组细胞在常规孵箱培养,缺氧/复氧组细胞利用缺氧孵箱和常规孵箱交替培养,山奈酚组细胞在缺氧/复氧培养的基础上应用30μmol/L山奈酚。分别利用流式细胞仪和多核苷酸链断裂技术检测各组细胞凋亡水平,利用蛋白印迹技术检测凋亡相关蛋白和钙离子通道蛋白含量,利用免疫荧光技术检测细胞中钙离子浓度。结果:和对照组比较,缺氧/复氧组细胞凋亡增加,钙离子通道蛋白Cav1.2表达和细胞中钙离子含量增加(均P<0.05);和缺氧/复氧组比较,山奈酚组细胞凋亡减少,钙离子通道蛋白Cav1.2表达和胞内钙离子浓度减少(均P<0.05)。结论:山奈酚可能通过减少钙离子通道蛋白Cav1.2表达和胞内钙离子浓度抑制缺氧/复氧损伤诱导的大鼠心肌细胞H9C2凋亡。

盐酸小檗碱对大鼠体内低剂量阿托伐他汀的药动学影响研究

目的:研究盐酸小檗碱对大鼠体内低剂量阿托伐他汀的药动学影响。方法:选取18只SD健康雄性大鼠进行研究,以随机双盲对照法将其分成A组、B组、C组,每组6只。其中A组予以10mg/kg的阿托伐他汀钙灌胃处理;B组予以小檗碱30mg/kg+阿托伐他汀钙10mg/kg相继灌胃;C组予以小檗碱90mg/kg+阿托伐他汀钙10mg/kg相继灌胃。检测并比较三组大鼠眼眶血中阿托伐他汀钙含量、血药浓度,绘制阿托伐他汀钙的药时曲线,计算药代动力学参数。此外,采集所有大鼠肝脏组织,检测细胞色素P450(CYP450)以及P-糖蛋白(P-gp)蛋白表达情况。比较各组大鼠肝脏组织UGT、GST及GSH含量。结果:B组、C组大鼠给药2h后血浆阿托伐他汀钙含量分别为(7.04±0.37)μg/ml、(7.88±0.43)μg/ml,均高于A组的(6.00±0.31)μg/ml,且C组大鼠给药2h后血浆阿托伐他汀钙含量高于B组(均P<0.05);三组大鼠给药前以及给药4h、6h、8h后的血浆阿托伐他汀钙含量对比均不明显(均P>0.05)。B组、C组大鼠t_(max)水平低于A组,且C组大鼠t_(max)水平低于B组;B组、C组大鼠C_(max)、t_(1/2)、CL以及MRT水平均高于A组,且C组大鼠C_(max)、t_(1/2)、CL以及MRT水平均高于B组(均P<0.05)。B组、C组肝脏CYP450以及P-gp表达均高于A组(均P<0.05);且B组和C组肝脏CYP450以及P-gp表达的差异无统计学意义(均P>0.05)。B组、C组大鼠肝组织UGT、GST活性均低于A组,且C组上述指标活性均低于B组;B组、C组大鼠肝组织GSH活性均高于A组,且C组GSH活性高于B组(均P<0.05)。结论:盐酸小檗碱对大鼠体内低剂量阿托伐他汀的药动学有一定影响,表现为血药浓度有升高趋势,需谨慎联用。

MicroRNA expression profile and lipid metabolism characteristics in liver of rat undergoing high-fat diet

This study aimed to investigate the microRNA expression profile and the characteristics of lipid metabolism in the livers of rats undergoing a high-fat diet.Fifty male Sprague-Dawley(SD)rats were divided into a standard chow group(C group,N=10)and a high-fat diet group(H group,N=40).After 12 weeks,the rat body weight,body length,fat mass,and serum lipid concentration were measured.The expression profile of microRNAs and the gene and protein expression levels involved in lipid metabolism in rat liver were detected.Body fat and serum lipid concentrations were all significantly higher in the H group than those in the C group(p<0.05 or p<0.01).The expression of 10 microRNAs showed significant differences in the liver(p<0.05).In particular,the let-7 family expression levels significantly increased(p<0.05)in the H group compared with those in the C group.Compared with the C group,the high-fat diet resulted in low FAS,CPT1A,and ApoAI mRNA expression levels(p<0.05 or p<0.01)and high PPARαand FAT/CD36 mRNA expression levels in the H group rat liver(p<0.01).Meanwhile,the protein PPARα,FAS,CPT1A,FAT/CD36,and ApoAI expression levels were all significantly lower in the H group than those in the C group(p<0.05 or p<0.01).In conclusion,the high-fat diet increased the body fat and serum lipid levels and altered the 10 microRNA expression levels in the liver.The high-fat diet may affect hepatic carbohydrate metabolism and increase ectopic fat accumulation through let-7 family overexpression.The high-fat diet for 12 weeks decreased lipid metabolism level in the liver,thereby decreasing fatty acid synthesis,oxidation,and transport by down-regulating the PPARα,FAS,CPT1A,FAT/CD36,and ApoAI protein levels.

Adrenocortical system activity in alloxan-resistant and alloxan-susceptible Wistar rats

In the dynamics of the disease development, diuresis and glycosuria increase in alloxan-susceptible rats, while in alloxan-resistant rats the increase in the values of these indices is expressed to a lesser extent, and they begin to decrease by day 8 of the disease. In alloxan-susceptible rats, the mass index of adrenal gland is increased, and that of thymus is decreased and corticosterone concentration in blood, adrenal gland and urine as well as alanine and aspartate aminotransferase activities in liver are increased;in alloxan-resistant rats the values of these indices do not differ from those of rats of the control group.

Evaluation of the Haematinic Activities of Extracts of <i>Justicia secunda</i>Vahl Leaves in Red Blood Cells of Laboratory Rats

The use of plant parts (leaves, flowers, stems, barks, roots etc.) in traditional medicine is increasingly gaining ground in modern medicine, as plant sources have long been recognized as sources of secondary metabolites which can be used to treat a wide range of diseases. The effect of extracts of Justicia secunda leaves on red blood cells (RBC) count and haemoglobin (Hb) concentration was investigated in adult Sprague-Dawley rats to establish haematinic activity. Phenylhydrazine (PHZ)-induced anaemic rats were treated with water, methanol or ethyl acetate extracts at 200 mg/kg body weight. RBC counts and Hb concentration were analysed using a haematology analyser at 3-day intervals for 21 days. The extracts were compared with rats administered the haematinic tonic Feroglobin&reg;?and vehicle-treated (normal saline). Rats administered the water extract exhibited the most significant increase (P ) in the number of RBCs and Hb concentration compared with the vehicle-treated PHZ-induced anaemic rats. Rats administered the methanol extract followed with significant increase (P ) in RBC counts and Hb concentration (J. secunda leaves has excellent haematinic properties and this provides the pharmacological basis of its use in Ghanaian traditional medicine for the treatment of anaemia.

Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation

INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but failed.We introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinalepithelial cell by electron microscopy,immunihistochemistry and enzymatic histoch-emistry. It has been applied to some relatedresearch work[17-21]. It was found that manyfactors were involved in the culture system. Ourpresent study focuses on the culture method and theinfluencing factors on IEC-6.

Qualitative Differences in Pup-Retrieval Strategies in a Maternal Separation Paradigm

The rodent maternal separation (MS) paradigm is frequently used to investigate the impact of early-life conditions in the offspring. One critical issue is whether the effects seen in the offspring are a result of maternal contact deprivation and/or altered pup-directed maternal behavior. To address this question we used an innovative approach with a qualitative analysis of pup-retrieval strategies in a test situation related to risk for the pups. The dams were separated from their litters for 0 (MS0) or 360 (MS360) min, respectively. The pups were placed in a risk area in the multivariate concentric square field? test at two test occasions and the pup-retrieval strategies were recorded. No significant evident differences between MS0 and MS360 dams were found. However, there were clearly two different strategies, either removing the pups out of potential danger or into safety, and these strategies were represented in both MS groups. As compared to the MS0 dams, the MS360 dams did not change their strategies and left more pups in the risk area in both pup-retrieval tests. This implies different pup-retrieval strategies depending on early-life conditions.

Teratogenic Effect of Paulownia Flower Extract on Rats

In this study, Paulownia flower extract of different concentrations was administered to pregnant rats by gavage to investigate its teratogenic effect. It was found that Paulownia has neither teratogenic effect nor growth inhibitory effect on rats, and it can be used in animal production as new feed additive.

不同剂量万古霉素对慢性心力衰竭大鼠心功能、 肾功能及神经内分泌因子的影响及其血药浓度变化

目的探讨不同剂量万古霉素对慢性心力衰竭(CHF)大鼠心功能、肾功能及神经内分泌因子的影响及其血药浓度变化。方法取12只大鼠作为假手术组。取60只大鼠建立CHF模型,将55只造模成功的大鼠随机分为CHF组(11只)、低剂量万古霉素组(11只)、中剂量万古霉素组(11只)、高剂量万古霉素组(11只)及药物对照组(11只)。分别给予低剂量万古霉素组、中剂量万古霉素组、高剂量万古霉素组大鼠经尾部注射25 mg/kg、50 mg/kg、100 mg/kg的万古霉素,给予药物对照组大鼠卡托普利灌胃,给予假手术组和CHF组大鼠等体积生理盐水灌胃。检测各组大鼠心功能指标[左心室舒张末期内径(LVEDD)、收缩末期内径(LVESD)、左心室射血分数(LVEF)和左室短轴缩短率(LVFS)]、肾功能指标[血清肌酐、血清尿素氮和24 h尿蛋白(UP24)]、神经内分泌因子[血清血管紧张素Ⅱ(AngⅡ)、醛固酮和心钠素]水平;采用HE染色观察各组大鼠心肌组织、肾组织的病理学变化;采用Western blot检测各组大鼠心肌组织AngⅡ、醛固酮和心钠素的蛋白表达水平;采用吸波面积法检测不同剂量万古霉素组的血药浓度。结果(1)与假手术组相比,CHF组大鼠的LVEDD、LVESD、血清肌酐水平、血清尿素氮水平和UP24水平均升高,LVEF、LVFS均降低(均P<0.05);与CHF组相比,各剂量万古霉素组大鼠的LVEDD、LVESD、血清肌酐水平、血清尿素氮水平和UP24水平均降低,LVEF、LVFS均升高(均P<0.05),除肾功能指标外,其余指标总体上均呈剂量依赖性变化。(2)HE染色结果显示,CHF组大鼠心肌细胞水肿,细胞浆染色加深,细胞体积缩小,可见空泡;肾小球间质增加,肾间质增宽。各药物干预组大鼠的心肌组织和肾脏组织损伤均有所减轻,其中高剂量万古霉素和药物对照组大鼠心肌损伤的改善最为显著,中剂量万古霉素组及药物对照组大鼠肾组织损伤的改善最为显著。(3)与假手术组相比,CHF组大鼠血清AngⅡ、醛固酮和心钠素水平,以及心肌组织AngⅡ、醛固酮和心钠素蛋白表达水平均升高(均P<0.05);与CHF组相比,各剂量万古霉素组大鼠血清AngⅡ、醛固酮和心钠素水平,以及心肌组织AngⅡ、醛固酮和心钠素蛋白表达水平均降低(均P<0.05),总体上呈剂量依赖性变化。(4)万古霉素血药浓度分析结果显示,低剂量万古霉素组的血药浓度在用药后48 h时达到峰值(8.16 mg/L),在用药后96 h时血药浓度为0;中剂量万古霉素组的血药浓度在用药后52 h时达到峰值(12.63 mg/L),在用药后144 h时血药浓度为0;高剂量万古霉素组的血药浓度在用药后72 h达到峰值(16.30 mg/L),在用药后96 h、122 h及148 h时血药浓度较稳定。结论不同剂量万古霉素均可改善CHF大鼠的心功能与肾功能指标,其作用机制可能与抑制神经内分泌因子AngⅡ、醛固酮和心钠素的表达水平有关;高剂量(100 mg/kg)万古霉素对CHF大鼠的心功能和心肌组织损伤的改善作用最佳,且血药浓度更为稳定,但对肾脏有一定损伤;中剂量(50 mg/kg)万古霉素对CHF大鼠的肾功能的改善作用最佳。

浓缩生长因子促进大鼠下颌骨联合部成骨作用的初步研究

目的探讨CGF对促进大鼠下颌骨联合部成骨的作用及机制研究。方法24只SD大鼠随机分为三组,于大鼠下颌骨联合部缺损处分别填满:单纯Bio-Oss胶原骨(胶原骨组);Bio-Oss胶原骨并于表面覆盖CGF凝胶(CGF+胶原骨组);单纯CGF凝胶(CGF组)。分组手术第3、6个月后,分两批处死大鼠。离体标本拍摄micro-CT,通过三维重建及截断面比较各组间放射学表现,按标准评分后进行统计学分析。标本拍摄micro-CT完成后脱钙处理,制作组织学切片,观察比较各组间的差异。结果结果表明第3个月时,CGF+胶原骨组评分要明显高于CGF组和胶原骨组(P=0.0266,P=0.0266),而CGF组和胶原骨组间差异无统计学意义;第6个月时,CGF+胶原骨组评分与CGF组和胶原骨组相比,具有显著优势(P=0.0021,P=0.0101),而胶原骨组评分看似优于CGF组,却差异无统计学意义。通过HE染色发现,CGF+胶原骨组在第3个月时评分显著优于CGF组和胶原骨组(P=0.0038,P=0.0038),而CGF组和胶原骨组间差异无统计学意义;第6个月时,与CGF组和胶原骨组相比,CGF+胶原骨组评分依然具有显著优势(P=0.0082,P=0.0194),胶原骨组评分略微高于CGF组,然而在统计学分析上无显著差异。结论CGF与胶原骨联合使用可以明显促进骨缺损的修复,但单独使用CGF对大范围骨缺损的效果并不理想。

不同频率电刺激对2型糖尿病大鼠血糖水平及骨骼肌卫星细胞激活的影响

目的:本文旨在探究不同电刺激参数对大鼠机体血糖调控和骨骼肌卫星细胞的激活效果。方法:通过比较不同参数条件的肌肉电刺激对2型糖尿病大鼠的空腹血糖浓度和口服葡萄糖耐量的改善效果,验证肌肉电刺激对2型糖尿病大鼠机体血糖调控的影响以及确定对大鼠血糖调控最有效的电刺激条件。以及对大鼠骨骼肌卫星细胞标志性蛋白Pax7、MyoD和MyoG进行免疫荧光染色,比较不同参数条件的肌肉电刺激对大鼠肌肉卫星细胞激活效果。结果:(1)20 Hz、40 Hz和100 Hz都对2型糖尿病大鼠的空腹血糖浓度和口服葡萄糖耐量具有改善作用,其中40 Hz电刺激改善效果最为明显。(2)100 Hz电刺激对大鼠肌卫星细胞的激活效果最为明显。结论:三种电刺激诱导肌肉自主收缩来控制机体血糖,其中,40 Hz电刺激改善效果最明显;三种刺激均可有效诱导肌卫星细胞的激活,100 Hz电刺激的激活效果最显著。

不同浓度二甲双胍对大鼠离体成骨细胞增殖和分化的影响

目的 观察不同浓度二甲双胍对大鼠离体成骨细胞增殖和分化的影响。方法 从SD乳鼠颅骨中分离出原代成骨细胞后进行培养和鉴定。用不同浓度(0μmol/L、50μmol/L、100μmol/L、200μmol/L、400μmol/L、800μmol/L、1 600μmol/L、3 200μmol/L)二甲双胍干预成骨细胞72 h后,检测成骨细胞的增殖情况。用不同浓度(0μmol/L、50μmol/L、200μmol/L、800μmol/L、1 600μmol/L、3 200μmol/L)二甲双胍干预成骨细胞72 h后,检测成骨细胞的碱性磷酸酶(ALP)活性,以及骨形成指标ALP、Ⅰ型胶原(COL-Ⅰ)的基因和蛋白表达水平。结果 (1)干预72 h后,与0μmol/L二甲双胍相比,经50μmol/L、100μmol/L、200μmol/L、400μmol/L、800μmol/L二甲双胍干预后成骨细胞的增殖活性呈浓度依赖性增强,其中经800μmol/L二甲双胍干预的成骨细胞增殖活性最强(P<0.05),而经3 200μmol/L二甲双胍干预后成骨细胞的增殖活性降低(P<0.05)。(2)在0~800μmol/L浓度范围时,二甲双胍可浓度依赖性地增强成骨细胞中ALP的活性,但1 600μmol/L及3 200μmol/L二甲双胍可抑制成骨细胞中ALP的活性。(3)与0μmol/L二甲双胍相比,经200μmol/L、800μmol/L二甲双胍干预后成骨细胞中ALP、COL-Ⅰ的mRNA和蛋白表达水平升高,而经3 200μmol/L二甲双胍干预后成骨细胞中ALP、COL-Ⅰ的mRNA表达水平及COL-Ⅰ的蛋白表达水平下降(P<0.05);在0~3 200μmol/L浓度范围内,二甲双胍浓度为800μmol/L时成骨细胞中ALP、COL-Ⅰ的mRNA和蛋白表达水平最高(P<0.05)。结论 不同浓度二甲双胍对成骨细胞影响不同,较低浓度(≤800μmol/L)二甲双胍可促进成骨细胞增殖和分化,较高浓度(3 200μmol/L)二甲双胍则抑制成骨细胞增殖和分化。

外源性碱性成纤维细胞生长因子对大鼠阴茎肌源性干细胞增殖的影响

目的探讨碱性成纤维细胞生长因子对大鼠阴茎肌源性干细胞体外增殖的影响。方法本试验自2020年12月至2021年6月在滕州市中心人民医院精准实验室完成。取健康雄性Wister大鼠5只,体质量(200±50)g,取其阴茎海绵体并采用酶消化法分离肌源性干细胞,应用密度梯度离心法和差速贴壁法进行纯化。取第2代肌源性干细胞分为两组进行培养,实验组:200μl生长培养基+肌源性干细胞,分别用终浓度为10μg/L、30μg/L、50μg/L、70μg/L、90μg/L外源性碱性成纤维细胞生长因子进行干预;对照组:加入200μl生长培养基+肌源性干细胞。分别于培养24 h、48 h、72 h、96 h、120 h、144 h加入四甲基偶氮唑蓝(MTT)溶液。应用免疫细胞化学法鉴定大鼠阴茎肌源性干细胞,MTT比色法检测细胞增殖情况。统计学方法采用独立样本t检验、Tukey检验。结果(1)肌源性干细胞多呈Sca-1阳性反应[表达量为(79.90±1.82)%],少数呈Desmin阳性反应[表达量为(68.60±0.72)%]。(2)两组肌源性干细胞均随培养时间的延长而明显增殖(均P<0.01),出现显著促增殖效应的培养时间均为96 h。(3)与对照组比较,实验组各浓度碱性成纤维细胞生长因子对肌源性干细胞均有明显的促增殖效应;当浓度从10~70μg/L递增时,促增殖作用也随之均显著增强(均P<0.01),至70μg/L时其促增殖作用接近顶峰。结论外源性碱性成纤维细胞生长因子可促进体外培养的大鼠阴茎肌源性干细胞增殖,且呈浓度-时间依赖性增加,96 h与70μg/L为最佳的体外培养时间和浓度。

两性霉素B制剂细胞毒性测定法

目的:建立一种能够在细胞水平快速评价两性霉素B制剂细胞毒性的方法,用于产品的质量研究。方法:以大鼠红细胞作为药物毒性评价载体,选择其红细胞中释放的K^(+)浓度为评价指标,以高效液相色谱法(HPLC)检测K^(+)浓度。结果:大鼠红细胞孵育时间为4 h时,组内样品平行性良好,批间阳性及阴性对照K^(+)释放结果重复性好,最大RSD为5.57%,符合质量要求。结论:本方法可以精确测定出不同剂型、不同浓度、不同孵育时间红细胞释放的K~+含量,并且不受其他杂质离子的干扰,可以用于快速评价两性霉素B制剂细胞毒性。

UPLC法测定大鼠血浆中Liguzinediol浓度以及动力学研究

目的建立测定大鼠血浆中Liguzinediol浓度的UPLC测定方法,探讨其在大鼠体内的药代动力学。方法血浆样品经甲醇提取后,上清液经N2吹干流动相复溶后注入UPLC分析,色谱柱为UPLC HSS T3柱(2.1 mm×100 mm,1.8μm),流动相为甲醇-水(28∶72,V∶V),流速为0.4 ml.min-1,检测波长278 nm。SD大鼠6只,iv给药10 mg.kg-1后,用UPLC法测定给药后大鼠血浆中Liguzinediol的浓度,利用DAS软件拟合并计算其药代动力学参数。结果 Ligu-zinediol的血药浓度在0.41～52.4 mg.L-1范围内呈线性,提取回收率均>80%,日内、日间精密度<10%,符合生物样品分析要求。大鼠静脉注射10 mg.kg-1的Liguzinediol,其血药浓度(C)-时间(t)曲线呈二室模型,主要药动学参数T12β、AUC(0-∞)、CL分别为1.62 h、18.36 mg.h.L-1、0.71 L.h-1.kg-1。结论该方法操作简便、快速、专属性强,可用于Liguzinediol的药代动力学及成药性研究。