外用重组人粒细胞-巨噬细胞刺激因子凝胶与注射用重组人生长激素联合应用对实验性烫伤动物的疗效

目的观察外用重组人粒细胞-巨噬细胞刺激因子(rhGM-CSF)凝胶与注射用重组人生长激素(rhGH)联合应用对实验性烫伤动物的疗效。方法将脱毛的Wistar大鼠背部皮肤浸入(85±1)℃恒温水浴中持续15s,造成深Ⅱ度烫伤。大鼠烫伤部位分别涂抹外用rhGM-CSF凝胶、易孚凝胶、美宝湿润烧伤膏、磺胺嘧啶银、医用凡士林膏,金因肽组用金因肽喷湿创面,rhGH组和GM+GH组在烫伤后24h开始皮下注射rhGH。各组每天给药1次,连续用药25d。每5d用透明硫酸纸描绘烫伤皮肤面积,并称重,计算烫伤皮肤愈合率;烫伤后6h、9d和25d,各组随机选取3只大鼠,对其烫伤创面进行病理学检查。结果从给药第10天开始,各给药组大鼠烫伤皮肤愈合率均比模型组明显提高;以GM+GH组最高。病理学观察显示,烫伤后25d,各给药组大鼠绝大多数创面表皮细胞增生明显,真皮层可见毛囊、皮脂腺腺体修复,GM+GH组炎细胞浸润不明显,模型组多见化脓性炎症改变,并见脓肿灶形成。结论各给药组均可减轻烫伤后炎症反应,促进烫伤皮肤愈合,对深Ⅱ度烫伤大鼠皮肤有明显治疗作用,其中以外用rhGM-CSF凝胶联合应用rhGH的治疗效果最好。     

注射用重组人B淋巴细胞刺激因子受体-抗体融合蛋白在恒河猴体内的安全性

目的评价注射用重组人B淋巴细胞刺激因子受体-抗体融合蛋白(RCT-18)在恒河猴体内的安全性。方法将恒河猴随机分为4组:低、中、高剂量RCT-18组和对照组,每组8只,低、中、高剂量RCT-18组分别经皮下注射4.6、16.1、57.5 mg/(kg.次)RCT-18,对照组经皮下注射0.7 ml/(kg.次)0.9%氯化钠注射液,每3 d上午同一时间给药1次,共给药91次。每天上午观察记录恒河猴的一般体征,每周常规监测体温1次,并在第1、2、3、13、14、15、28、29和30次给药前、给药后0.5、1、2、4和24 h增加测量动物体温次数。给药0.5、1.5、3、4.5、6、7.5、9个月(停药次日)及停药48 d(恢复期结束),分别对心电图、眼科、血液学、血液生化学、尿常规、血清抗RCT-18抗体、外周血单个核细胞(PBMC)分类计数、血清免疫球蛋白水平(IgG、IgA和IgM)等各项指标进行检查。给药3、6、9个月和恢复期结束,每组各解剖2只猴,进行脏器大体观察,计算脏器系数,并进行组织病理学观察。结果临床反应主要表现为给药后的体温升高;血液学指标出现具有一定剂量-反应关系的单核细胞(monocyte,MONO)数和网织红细胞(reticulocyte,RETIC)升高或网织红细胞平均体积(mean reticulocyte volume,MCVr)降低;3个剂量RCT-18组恒河猴的脏器重量和脏器系数与同期对照组相比,均无明显变化,且未见明显的脏器大体病变;3个剂量RCT-18组均出现无明显剂量相关但可逆的脾小结和淋巴滤泡萎缩;在整个试验期内未检测出血清中的抗RCT-18抗体;3个剂量组外周血淋巴细胞中IgD+细胞(成熟B淋巴细胞)比率均略有下降,但与对照组相比,差异无统计学意义(P>0.05);血清IgG、IgA和IgM从给药0.5～1.5个月后开始明显下降,恢复期结束时,除了中剂量的IgA水平,其他3个剂量RCT-18组的IgG、IgA和IgM水平均出现不同程度的回升。结论 RCT-18对恒河猴具有明显的但可恢复的免疫抑制作用。     

人用重组禽流感血凝素疫苗接种大鼠的安全性及免疫原性

目的评价家蚕生物反应器生产的重组禽流感血凝素疫苗的安全性和免疫原性。方法将SD大鼠随机分为5组,分别经皮下注射9.4、1.88和0.375mg/kg剂量的重组禽流感血凝素疫苗(含氢氧化铝佐剂)、A(lOH)3和生理盐水,每10d注射1次,连续注射3次,分别于第1次给药后第2、22和42天进行血液学、血液生化学、病理学和免疫原性检测。结果3个剂量组疫苗第1次给药后第42天,检测抗体滴度(P/N)在8～11之间,CD4+、CD8+T淋巴细胞含量及IFNγ和IL-2的含量与两对照组相比,未见明显升高;连续3次给药后,大鼠的血液学和生化学指标均无明显改变;给药期间注射部位皮下出现肉芽肿样结节,经20d恢复后有所改善;9.4和1.88mg/kg剂量组动物出现可逆性的脾肿大症状;整个实验期间,动物未出现任何其他明显不良反应。结论重组禽流感血凝素疫苗对大鼠具有良好的安全性,但免疫原性较低。     

西红花苷联合叶黄素对糖尿病大鼠视网膜PEDF表达的影响

目的:通过大鼠视网膜色素上皮衍生因子(PEDF)模型评价联合使用西红花苷和叶黄素对视网膜的保护作用。方法:采用链脲佐菌素(65 mg/kg)一次性ip SD大鼠建立糖尿病大鼠模型,给药组分别为西红花苷、叶黄素和西红花苷叶黄素(1∶1)。20周后处死动物,检测血清PEDF;摘除眼球应用免疫组织化学观察PEDF蛋白的表达,同时取视网膜,采用荧光定量PCR法检测视网膜PEDFmRNA的表达。结果:西红花苷组、叶黄素组和联合用药组血清PEDF均比模型组高,联合用药组血清PEDF显著高于红花苷和叶黄素单独用药组(P0.05),此外,西红花苷组、叶黄素组和联合用药组PEDFmRNA表达均比模型组高,联合用药组PEDFmRNA表达显著高于西红花苷和叶黄素单独用药组(P0.05)。结论:西红花苷叶黄素联合使用通过促进PEDF的表达,从而对糖尿病的视网膜组织有协同增强疗效的保护作用。     

新生鼠提取液对急性血瘀模型大鼠血液流变学的影响

目的　探讨新生鼠提取液 (NRE)对急性血瘀模型大鼠血液流变学的影响。方法　大鼠皮下注射0 1%肾上腺素 0 2ml后放冰水中浸泡 5min复制急性血瘀模型 ,应用NRE进行治疗 (NRE 2 0和 4 0ml kg每天灌胃给药 1次 ,连续 7d) ,测定大鼠血液流变学各参数变化。结果　急性血瘀模型大鼠全血黏度、血浆黏度、血浆纤维蛋白原浓度、血沉、红细胞聚集指数及红细胞刚性指数均明显降低 ,红细胞电泳时间及红细胞压积则无明显影响。结论　NRE可明显改善急性血瘀模型大鼠血液流变学 ,对于防止动脉硬化的发展及并发症的发生均有益处。     

BCG-CpG-DNA一般毒性的初步评价

目的观察BCG-CpG-DNA对小鼠急性毒性和28d长期毒性作用,评价其一般毒性。方法小鼠分别经背部皮下给予BCG-CpG-DNA100、250和500μg1次(急性毒性试验)和7次(28d长期毒性试验),对照组注射生理盐水。单次给药后观察小鼠的生长情况和体重变化,14d后观察脏器是否发生病变;重复给药期间及末次给药后3周恢复期内,每周观察小鼠外观体征、局部刺激性和体重变化;末次给药后3d及3周检测血液学、血液生化学指标及主要脏器系数和抗双链DNA抗体水平。结果 BCG-CpG-DNA急性毒性试验中,各组小鼠均健康存活,体重增加,无毒性反应。BCG-CpG-DNA28d长期毒性试验中,动物体重、行为活动无异常,给药部位无局部刺激性表现;BCG-CpG-DNA重复给药可提高小鼠免疫细胞的数量;末次给药后3d,各试验组脾脏系数均高于对照组,且差异有统计学意义(P<0.05),经过3周恢复期回复正常;各试验组血清中抗双链DNA水平与对照组比较,差异无统计学意义,均低于25U/ml的临界值。结论 BCG-CpG-DNA主要影响小鼠的免疫细胞和免疫器官,未见其他明显的急性毒性和长期毒性作用,表明BCG-CpG-DNA具有较好的安全性。     

重组人红细胞生成素对慢性肾衰大鼠的疗效

建立慢性肾衰贫血大鼠的动物模型后 ,分别用基因重组人红细胞生成素 (r Hu EPO)按 45 0 IU /kg、15 0 IU /kg、 5 0 IU /kg 3组剂量连续 14d皮下注射上述大鼠 ,同时设立阳性对照组及阴性对照组。结果发现r Hu EPO只能治疗肾衰造成的贫血 ,而对肾衰无治疗作用。     

腺嘌呤致大鼠慢性肾功能衰竭贫血动物模型的建立

给大鼠口服含 0 .75 %腺嘌呤饲料 7周 ,造成大鼠慢性肾功能衰竭 ,并引起较严重的贫血 ,为进行重组人红细胞生成素 (rHuEPO)对慢性肾衰所致贫血的药效研究提供了稳定的实验动物模型。     

艾纳香口腔护理液安全性评价研究

选用SD大鼠进行口腔黏膜刺激性试验、口腔及黏膜用药急性毒性试验;选用白色豚鼠进行过敏试验;选用昆明种小鼠进行灌胃给药急性毒性试验,观察给药后动物口腔黏膜、受药皮肤、体重变化、全身毒性反应情况及死亡只数。艾纳香口腔护理液在口腔黏膜刺激性试验中,未发现SD大鼠口腔黏膜组织异常;在过敏试验中,白色豚鼠皮肤用药未引起过敏反应;在急性毒性试验中,大鼠的耐受性良好;小鼠灌胃时测得的最大给药量(MLD)为288 mg/kg,相对于成人给药量的1 200倍。上述动物均未见死亡和明显毒性反应。以上试验说明艾纳香口腔护理液无口腔黏膜刺激性和致敏性,无明显急性毒性反应,提示其作为口腔外用护理液临床用药安全。     

注射用重组人B淋巴细胞刺激因子受体-抗体融合蛋白对大鼠生育力及早期胚胎发育的毒性作用

目的观察注射用重组人B淋巴细胞刺激因子受体-抗体融合蛋白(RCT-18)对大鼠生育力及早期胚胎发育的毒性作用。方法将SD大鼠随机分为4组,分别经皮下注射RCT-18 129、37、11 mg/(kg.d)和0.9%NaCl注射液,每2 d给药1次。雄鼠连续给药5周、雌鼠连续给药2周后,按雌雄1∶1的比例合笼交配,合笼期为2周,计算交配率。雄鼠继续给药至交配成功,雌鼠继续给药至妊娠第6天。实验过程中观察动物的一般反应、体重及摄食量变化。确定雌鼠受精后处死雄鼠,解剖检查睾丸和附睾等生殖器官。于妊娠第15天解剖孕鼠,综合评价妊娠及胚胎形成和早期发育等情况。结果 RCT-18高、中、低剂量组雌性和雄性大鼠均未出现明显的母体毒性反应。各组大鼠各脏器均未见肉眼可见的异常。各剂量RCT-18对大鼠的交配率、妊娠率、生殖系统脏器重量和系数以及早期胚胎发育均无明显影响。结论RCT-18对SD大鼠的生育力及早期胚胎发育无明显毒性作用。     

重组人干扰素β1b治疗复发缓解型多发性硬化症的安全性及临床疗效

目的观察重组人干扰素β1b(rhIFNβ1b)治疗复发缓解型多发性硬化症(RRMS)的安全性及临床疗效。方法选择20名健康志愿者,分为1.5、3、6和9MIU4个剂量组,每天按剂量皮下注射1次rhIFNβ1b,连续注射8d,观察药物的安全性、耐受性和药代动力学。选择101例RRMS患者,随机分为观察组(52例)和对照组(49例),每周3次分别注射6MIU的rhIFNβ1b和25mg人血白蛋白,用药1年后,2组均注射6MIU的rhIFNβ1b,每周3次,用药1年。定期随访并进行各项指标及核磁共振成像(MRI)检查,观察药物的临床疗效,同时进一步观察安全性。结果安全性观察显示,rhIFNβ1b在9MIU剂量范围内耐受性良好。观察组和对照组分别注射rhIFNβ1b后,可显著减少RRMS患者头部与脊髓中病灶数量或缩小病灶体积,并显著降低复发率,且随着用药时间的延长,效果更加明显。结论rhIFNβ1b安全性和耐受性良好,临床影象学评价疗效显著。     

Spinal Stroke: Outcome Attenuation by Erythropoietin and Carbamylated Erythropoietin and Its Prediction by Sphingosine-1-Phosphate Serum Levels in Mice

Spinal strokes may be associated with tremendous spinal cord injury. Erythropoietin (EPO) improves the neurological outcome of animals after spinal cord ischemia (SCI) and its effects on ischemia-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are considered possible molecular mechanisms. Furthermore, sphingosin-1-phosphate (S1P) is suggested to correlate with SCI. In this study, the effect of recombinant human EPO (rhEPO) and carbamylated EPO (cEPO-Fc) on the outcome of mice after SCI and a prognostic value of S1P were investigated. SCI was induced in 12-month-old male mice by thoracic aortal cross-clamping after administration of rhEPO, cEPO-Fc, or a control. The locomotory behavior of mice was evaluated by the Basso mouse scale and S1P serum levels were measured by liquid chromatography-tandem mass spectrometry. The spinal cord was examined histologically and the expressions of key UPR proteins (ATF6, PERK, and IRE1a, caspase-12) were analyzed utilizing immunohistochemistry and real-time quantitative polymerase chain reaction. RhEPO and cEPO-Fc significantly improved outcomes after SCI. The expression of caspase-12 significantly increased in the control group within the first 24 h of reperfusion. Animals with better locomotory behavior had significantly higher serum levels of S1P. Our data indicate that rhEPO and cEPO-Fc have protective effects on the clinical outcome and neuronal tissue of mice after SCI and that the ER is involved in the molecular mechanisms. Moreover, serum S1P may predict the severity of impairment after SCI.     

Purification and characterization of recombinant human erythropoietin from milk of transgenic pigs

BACKGROUND: Human erythropoietin (hEPO), a hydrophobic acidic glycoprotein responsible for the regulation of red blood cell production in mammals, is used for the treatment of anemia. In general, the purification of transgenic animal‐derived therapeutic proteins is not easy due to their low titer concentrations and abundant contaminant proteins. For the first time, here the purification and characterization of rhEPO from the milk of transgenic pigs are described. RESULTS: The rhEPO was purified by heparin chromatography, reverse‐phase chromatography, and gel filtration chromatography, resulting in a 16.5% yield and > 98% purity. The rhEPO purified from the milk of transgenic pigs contained less acidic isoforms and was underglycosylated in contrast to CHO‐derived rhEPO. Cell proliferation of the F‐36/EPO‐dependent cell line was proportional to the dose of transgenic pig‐derived rhEPO. CONCLUSION: Transgenic pig‐derived rhEPO with high purity was achieved after three‐step chromatography following two‐step precipitation. The transgenic pig‐derived rhEPO was demonstrated to have comparable potency with CHO‐derived rhEPO. Transgenic pig‐derived rhEPO may not be therapeutically feasible because of different glycosylation, and thus further studies are required to elucidate the effect of this aberrant glycosylation on the biological activity and stability in vivo. Copyright © 2008 Society of Chemical Industry     

CpG-ODN对肾小球肾炎进展的影响

目的观察CpG-ODN对肾小球肾炎进展的影响。方法将Wistar雄性大鼠随机分为N组(正常对照)、M组(模型对照)、CpG组和GpC组。N组大鼠经尾静脉注射生理盐水2.5ml/(kg·d),其余3组大鼠经尾静脉注射抗-thy1.1单克隆抗体2.5ml/(kg·d),隔日1次,共3次。注射抗-thy1.1单克隆抗体后,隔天,CpG组经尾静脉注射CpG-ODN,300μg/只,GpC组经尾静脉注射GpC-ODN,300μg/只,隔日注射,共3次。各组大鼠分别于用药前和第1针注射后1周测定尿蛋白;用药后第8周留取大鼠24h尿、血清及肾脏组织,检测24h尿蛋白定量、血清白蛋白和肾功;肾脏组织用于肾脏病理检查以及RT-PCR法检测TLR-9mRNA的表达。结果CpG组与M组和GpC组相比,血清白蛋白含量明显降低,且差异有显著意义;M组、CpG组和GpC组在给药后1周均出现尿蛋白,在给药后8周,24h尿蛋白含量明显增多;CpG组与M组相比,TLR-9mRNA在肾脏的表达水平明显增加,且差异有显著意义;GpC组与M组相比,差异无显著意义;光镜下可见CpG组肾组织病理改变明显加重。结论天然CpG-ODN可促进肾小球肾炎病情加重,TLR-9可能在其发生机理中起重要作用。     

Randomized Controlled Trial of Darbepoetin α Versus Continuous Erythropoietin Receptor Activator Injected Subcutaneously Once Every Four Weeks in Patients with Chronic Kidney Disease at the Pre-Dialysis Stage

Continuous erythropoietin receptor activator (CERA) seems to maintain a stable hemoglobin (Hb) level because its half-life is longer than darbepoetin α (DA). Twenty chronic kidney disease (CKD) patients at the pre-dialysis stage who had been administered DA for over 24 weeks were randomly assigned to receive subcutaneous CERA or DA once every four weeks during 48 weeks. In both groups, the rate of achievement of target Hb level changed from 70% to 100% in weeks 0 to 48, with no significant difference between the groups. Compared with week 0, the Hb level was significantly increased from week 24 in the DA group and from week 8 in the CERA group. In addition, the reticulocyte count was significantly increased from week 4 in the CERA group compared with the DA group. There was no significant difference in the levels of estimated glomerular filtration rate and iron status between both groups. Because of the small number of patients in this study, only limited conclusions can be drawn. However, the results suggest that subcutaneous administration of DA or CERA once every four weeks to predialysis patients has similar effects on achievement of target Hb levels.     

循环应用低剂量环磷酰胺对黑色素瘤荷瘤小鼠调节性T细胞的影响及抗瘤作用

目的观察循环应用低剂量环磷酰胺(CTX)对黑色素瘤荷瘤小鼠调节性T细胞(Treg)的影响及抗瘤作用。方法通过皮下接种黑色素瘤细胞B16制备黑色素瘤荷瘤小鼠模型;对荷瘤小鼠分别经腹腔单次或循环注射CTX,每隔7d给药1次,共3次,应用流式细胞术检测小鼠脾脏中CD4+CD25+Foxp3+Treg的含量;培养小鼠骨髓来源的树突状细胞(DC),将DC与脾T淋巴细胞混合培养,应用ELISA法检测脾T淋巴细胞干扰素γ(IFNγ)的分泌量;同时测量肿瘤的大小,绘制肿瘤生长曲线,并观察各组荷瘤小鼠自身免疫病的发生情况。结果CTX的最佳应用剂量为100mg/kg。随着荷瘤时间的延长,荷瘤小鼠脾脏CD4+CD25+Foxp3+/CD4+的比值呈逐渐升高趋势。单次应用CTX抑制Treg的时间较短,而循环应用CTX能够延长对Treg的抑制,使其维持在相对较低的水平;循环应用CTX显著提高了荷瘤小鼠脾T淋巴细胞IFNγ的分泌水平。单次和循环应用CTX均未能延缓肿瘤的生长,两组小鼠均未见免疫性白斑及明显的化疗毒副反应。结论循环应用CTX能更加有效地调控Treg,从而促进DC对T淋巴细胞的抗原特异性激活,这将会提高DC疫苗的抗瘤效果。     

Cyclophilin Inhibition Protects Against Experimental Acute Kidney Injury and Renal Interstitial Fibrosis

Cyclophilins have important homeostatic roles, but following tissue injury, cyclophilin A (CypA) can promote leukocyte recruitment and inflammation, while CypD can facilitate mitochondrial-dependent cell death. This study investigated the therapeutic potential of a selective cyclophilin inhibitor (GS-642362), which does not block calcineurin function, in mouse models of tubular cell necrosis and renal fibrosis. Mice underwent bilateral renal ischemia/reperfusion injury (IRI) and were killed 24 h later: treatment with 10 or 30 mg/kg/BID GS-642362 (or vehicle) began 1 h before surgery. In the second model, mice underwent unilateral ureteric obstruction (UUO) surgery and were killed 7 days later; treatment with 10 or 30 mg/kg/BID GS-642362 (or vehicle) began 1 h before surgery. GS-642362 treatment gave a profound and dose-dependent protection from acute renal failure in the IRI model. This protection was associated with reduced tubular cell death, including a dramatic reduction in neutrophil infiltration. In the UUO model, GS-642362 treatment significantly reduced tubular cell death, macrophage infiltration, and renal fibrosis. This protective effect was independent of the upregulation of IL-2 and activation of the stress-activated protein kinases (p38 and JNK). In conclusion, GS-642362 was effective in suppressing both acute kidney injury and renal fibrosis. These findings support further investigation of cyclophilin blockade in other types of acute and chronic kidney disease.     

Maternal anemia in Concepción province, Chile: association with maternal nutritional status and fetal growth

Previous studies in Santiago, Chile have established that anemia in the earliest stages of pregnancy is a public health issue. The situation in other parts of the country is unknown. The purpose of this study is to establish the prevalence of anemia in pregnant women in the province of Concepcion and evaluate its association with maternal nutricional status and fetal growth. The study included 1782 women with singleton pregnancies who began prenatal check-ups in 2004 at the public primary health care centers. Anemia was established using the following criteria: from WHO (Hb < 11 g / dl) and from the USA Center for Disease Control (CDC) (Hb < percentile 5 for each gestational week). Anemia prevalence was compared in relation to independent study variables: maternal age, parity, morbidity and smoking habit, and mother and child anthropometry. A multivariable logistic regression model studied the possible effect of anemia on fetal growth. The prevalence of anemia was 10.9% and 14.5% using the WHO and CDC criteria, respectively. The mother's nutritional status was significantly associated with anemia. However, anemia according to WHO and CDC criteria at the beginning of pregnancy was not significantly associated to fetal growth in the univariate and multivariate analyses. The prevalence of anemia in the province of Concepcion constitutes a public health problem that needs to be addressed and it is slightly higher to that recently observed in the county of Puente Alto, Santiago.     

Therapeutic Effects of Acer palmatum Thumb. Leaf Extract (KIOM-2015E) on Benzalkonium Chloride-Induced Dry Eye in a Mouse Model

We determined the effects of two extracts from Acer palmatum Thumb. leaves (hot water extract KIOM-2015EW and 25% ethanol extract KIOM-2015EE) in a benzalkonium chloride (BAC)-induced dry eye mouse model. Dry eye was induced by 0.2% BAC for 2 weeks, followed by treatment three times (eye drop) or once (oral administration) daily with KIOM-2015E for 2 weeks. Treatment with both KIOM-2015EE and KIOM-2015EW resulted in a marked increase in tear volume production for the 4 days of treatment. The Lissamine Green staining score, TUNEL-positive cells, and inflammatory index were significantly decreased after 2 weeks. Topical KIOM-2015EE administration exhibited a greater improvement in decreasing the ocular surface staining scores, inflammation, dead cells, and increasing tear production in a dose-dependent manner compared with the other groups. Furthermore, KIOM-2015E significantly reduced the phosphorylation of NF-κB, which was activated in the BAC-treated cornea. Topical administration was much more effective than oral administration for KIOM-2015E and KIOM-2015EE was more effective than KIOM-2015EW. Application of KIOM-2015E resulted in clinical improvement, inhibited the inflammatory response, and alleviated signs of dry eye. These results indicate that KIOM-2015E has potential as a therapeutic agent for the clinical treatment of dry eye.