The research of degradability of a novel biodegradable coralline hydroxyapatite after implanted into rabbit

To examine the biodegradability and bone healing effect of a novel biodegradable coralline hydroxyapatite after implanting into the proximal tibia of rabbit. Seventy New Zealand white rabbits were enrolled, bone defects about 10 x 5 x 3 mm(3) of bilateral proximal tibias were prepared by drilling, then coralline hydroxyapatite and iliac crest bone were grafted into bilateral bone defects, respectively. Each time five rabbits were sacrificed at 1, 2, 3, 4, 6, 8, 10, 12, 20, 24, 32, 36, 40, and 60 weeks after surgery. Then a series of examination were carried out, including eye view, roentgenographically, and nondecalcification histological examination. Eye view and roentgenographical examination indicate that all the defects grafted with coralline hydroxyapatite exhibited bone fusion, similar to the iliac crest autograft. The bone density of the graft site decreases with time on the X-ray film. Nondecalcification histological examination results are as followed: In the early time on the sections, the coralline hydroxyapatite looks like interlinked trabecula. Few lymphocytes infiltrate around the trabecula. With time extending, coralline hydroxyapatite looks like thin line or thin circle remnant. The degradation sites are filled with renascence bone. Medulla cavity can be seen in the degradation sites. After grafted in body, coralline hydroxyapatite exhibits little local and general abnormal reaction. It conducts good bone fusion of fracture. Coralline hydroxyapatite can be degraded after grafted into body, which is good for remodeling of bone healing. Hence coralline hydroxyapatite is an ideal bone graft substitute of autograft.     

Antimycobacterial activity of Pichia pastoris-derived mature bovine neutrophil β-defensins 5

Tuberculosis (TB) is an ongoing threat to global health, and the lack of effective therapies for treating it is also a global problem. Previous studies have shown that human cathelicidin and defensins have effective antimicrobial activity against Mycobacterium spp. To our knowledge, there are no reports on the antimycobacterial effects of bovine neutrophil β-defensins so far. Here, we identified the antimicrobial effect of mature bovine neutrophil β-defensins (mBNBD) 5 against Mycobacterium infection both in vitro and in vivo. The mBNBD5 protein was expressed in Pichia pastoris. To increase the yield of β-defensins, a purification method was employed by adding a 6-His·tag to the C-terminus of the mBNBD5 gene. Our results indicated that recombinant mBNBD5 protein was successfully expressed and purified from Pichia pastoris with intact antimicrobial activity. The recombinant protein exhibited potent bactericidal activity in vitro against M. smegmatis and M. bovis, with a dose-dependent manner and a time-dependent manner. The electron microscope results showed that the bacterial cell wall of M. bovis was disrupted when incubated with mBNBD5 for 72 h. Our data also indicated that the exogenous addition of mBNBD5 could reduce the survival of Mycobacterium spp., especially M. tuberculosis and M. bovis in RAW 264.7 macrophages. These results provide foundations for the development of mBNBD5 as a potential new therapeutic agent for TB treatment.     

Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression in macrophage cells

Objective and design

Microgravity environments in space can cause major abnormalities in human physiology, including decreased immunity. The underlying mechanisms of microgravity-induced inflammatory defects in macrophages are unclear.

Material or subjects

RAW264.7 cells and primary mouse macrophages were used in the present study. Lipopolysaccharide (LPS)-induced cytokine expression in mouse macrophages was detected under either simulated microgravity or 1g control.

Methods

Freshly isolated primary mouse macrophages and RAW264.7 cells were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1g control conditions. The cytokine expression was determined by real-time PCR and ELISA assays. Western blots were used to investigate the related intracellular signals.

Results

LPS-induced tumor necrosis factor-α (TNF-α) expression, but not interleukin-1β expression, in mouse macrophages was significantly suppressed under simulated microgravity. The molecular mechanism studies showed that LPS-induced intracellular signal transduction including phosphorylation of IKK and JNK and nuclear translocation of NF-κB in macrophages was identical under normal gravity and simulated microgravity. Furthermore, TNF-α mRNA stability did not decrease under simulated microgravity. Finally, we found that heat shock factor-1 (HSF1), a known repressor of TNF-α promoter, was markedly activated under simulated microgravity.

Conclusions

Short-term treatment with microgravity caused significantly decreased TNF-α production. Microgravity-activated HSF1 may contribute to the decreased TNF-α expression in macrophages directly caused by microgravity, while the LPS-induced NF-κB pathway is resistant to microgravity.     

含有H5N1 NA基因重组腺病毒疫苗在小鼠体内的免疫效果研究

目的 考察含有禽流感病毒A/Anhui/1/2005(H5N1)优化型NA基因的重组腺病毒在BALB/c小鼠体内的免疫效果,并筛选合适的免疫剂量.方法 重组病毒以肌内注射方式在第0周和第4周免疫BALB/c小鼠两次,低、中、高组的免疫剂量分别是10~5、10~7和10~9 TCID_(50)/次,第5周时分别使用神经氨酸酶活性抑制试验和EHSpot实验来检测和比较疫苗的体液和细胞免疫效果.结果 重组病毒免疫后的小鼠均可检测出针对NA抗原的特异性体液和细胞免疫反应,并且免疫效果与免疫剂量呈正相关,10~7 TCID_(50)/只/次是合适的免疫剂量.另外,从包含NA全长氨基酸残基的合成肽库中筛选到两个能够刺激BALB/c小鼠T淋巴细胞分泌IFN-γ的表位,即NA_(109-124):CRTFFLTQGALLNDKH和NA_(182-199):AVAVLKYNGIITIKSW.结论 含有优化型NA基因的重组腺病毒疫苗能够诱导BALB/c小鼠同时产生NA抗原特异性的体液和细胞免疫反应,是较好的H5N1流感病毒候选疫苗株,值得进一步深入研究.     

双胎输血综合征选择性胎儿镜激光凝固手术前后胎儿心肌力学的研究

目的 探讨双胎输血综合征(TTTS)选择性胎儿镜激光凝固术(SFLP)前后供血胎儿和受血胎儿的心肌力学改变.方法 2007年10月至2010年3月对25例TTTS孕妇行SFLP,手术前24h和手术后1周行胎儿超声心动图检查,并采集胎儿二维标准四腔心数字化图像,用速度矢量成像(VVI)软件分析心脏左室和右室长轴方向的心肌应变、收缩期应变率(SRs)和舒张期应变率(SRd).结果 与术前比较,SFLP术后,供血胎儿心胸比增大(分别为0.29±0.03、0.34±0.05,P＜0.01),出现三尖瓣反流(7例)和心包积液(5例),心肌力学方面:左室和右室的心肌应变、SRs和SRd均明显减低[左室心肌应变:(-19.24±3.68)％、(-13.78±3.64)％,P＜0.01;左室SRs:(-2.28±0.53)、(-1.43±0.41)s-1,P＜0.01;左室SRd:(1.67±0.43)、(1.15±0.70) s-1,P＜0.01;右室心肌应变:(-20.20±3.19)％、(- 16.10±3.07)％,P＜0.01;右室SRs:(-2.03 ±0.65)、(-1.72±0.38) s-1,P＜0.05;右室SRd:(1.71±0.30)、(1.50±0.36) s-1,P＜0.05];而受血胎儿心胸比减小(分别为0.42±0.04、0.37±0.04,P＜0.01),左室和右室的心肌应变、SRs和SRd均明显升高[左室心肌应变:(-10.62±2.72)％、(- 16.46±3.23)％,左室SRs:(-1.09±0.30)、(-1.60±0.31)s-1,左室SRd:(0.99±0.34)、(1.53±0.32) s-1,右室心肌应变:(- 11.66±4.56)％、(- 17.96±3.97)％,右室SRs:(- 1.26±0.39)、(- 1.74±0.45)s-1,右室SRd:(1.15±0.49)、(1.63±0.44)s-1;P＜均0.01].结论 SFLP术后短时间内(1周),受血胎儿的心功能明显得到改善,而供血胎儿心功能则减退.     

三磷酸腺苷-肿瘤体外药敏试验联合耐药基因检测预测原发性卵巢癌的化疗敏感性

目的 评价三磷酸腺苷-肿瘤体外药敏试验(ATP-TCA)联合耐药基因检测预测原发性卵巢癌的化疗敏感性,为临床治疗提供参考.方法 选择2008-2009年间收治的接受肿瘤细胞减灭术的原发性卵巢癌患者47例,采用ATP-TCA检测卵巢癌患者对顺铂、卡铂、紫杉醇、紫杉醇+卡铂4种方案的体外药物敏感性,采用实时逆转录(RT)PCR技术检测其中46例(1份冻存的癌组织标本因降解较严重未予检测mRNA)卵巢癌患者的癌组织中耐药基因--BRCA1、ERCC1 mRNA的表达水平.采用x2检验分析ATP-TCA检测结果 与卵巢癌患者临床病理指标的相关性;采用ATP-TCA联合耐药基因检测预测卵巢癌患者对紫杉醇+卡铂方案的化疗敏感性.结果 (1)卡铂、顺铂、紫杉醇+卡铂3种方案的ATP-TCA检测结果 与临床疗效均有明显的相关关系(P<0.05),其中紫杉醇+卡铂方案的ATP-TCA检测结果 与临床疗效的符合率最高,达83%(39/47);而紫杉醇方案的ATP-TCA检测结果 与临床疗效无明显相关关系(P>0.05).紫杉醇+卡铂方案的ATP-TCA检测结果 预测临床疗效的灵敏度、特异度、阳性预测值、阴性预测值分别为90%、71%、84%、80%.紫杉醇+卡铂方案的ATP-TCA检测结果 与残瘤灶直径(P=0.023)、有无新辅助化疗(P=0.011)有明显相关关系.(2)临床化疗敏感和耐药患者BRCA1 mRNA的表达水平分别为0.673±2.143和-1.436±2.594,两者比较,差异有统计学意义(P=0.008);ERCC1 mRNA的表达水平分别为-0.529±1.982和-3.188±2.601,两者比较,差异也有统计学意义(P=0.001);BRCA1和ERCC1 mRNA的表达与临床疗效有明显的相关关系(P<0.01).(3)ATP-TCA联合耐药基因检测,预测临床应用紫杉醇+卡铂方案可能会发生耐药的准确率达到8/9.结论 ATP-TCA是一种比较理想的体外药敏检测方法,可以有效地预测临床化疗敏感性;ATP-TCA联合耐药基因检测可以提高预测卵巢癌临床化疗敏感性的准确率,指导卵巢癌的个体化治疗.

Abstract：

Objective To predict clinical chemotherapy sensitivity of primary ovarian cancer by jointing adenosine triphosphate(ATP) - tumor chemo-sensitivity assay(TCA) method in vitro and detection of drug resistance genes, provide reference for clinical treatment. Methods Forty-seven primary epithelial ovarian tumor samples were collected from the patients who received cytoreductive surgery. Viable ovarian cancer cells obtained from malignant tissue were tested for their sensitivity to carboplatin (CBP), cisplatin (DDP), paclitaxel(PTX) and CBP + PTX using ATP-TCA method in vitro; at same time, real-time quantitative PCR was used to analysis BRCA1 and ERCC1 mRNA relative expression in forty-six specimens (1 frozen tumor samples mRNA were not detected due to serious degradation). The relationship between ATP-TCA test results, clinical indicators, and the effectiveness of the joint prediction on clinical chemosensitivity by combining these two methods were statistically analyzed using chi-square test. Results (1)The results showns that three programs of DDP,CBP and PTX + CBP were significantly related with clinical results(P<0.05) in vitro, in which the compliance rate in PTX + CBP program was the highest 83%(39/47) ,and the predictive sensitivity, predictive specificity, positive predictive value, negative predictive value and predictive accurate rate were 90%,71%,84% and 80% ,respectively.PTX + CBP combined in vitro test results was also related with residual tumor size and neoadjuvant chemotherapy, which was more prone to drug resistance with residual tumor larger than 2 cm (P = 0. 023) and with neoadjuvant chemotherapy (P = 0.011). (2) BRCA1 mRNA expression levels in the clinical-resistant group and the clinical-sensitive group was 0.673 ± 2.143 and - 1.436 ± 2.594 (P=0.008), ERCC1 mRNA expression levels in the clinical-resistant group and the clinical-sensitive group was -0.529 ± 1.982 and - 3.188 ±2.601 (P =0.001). There were also significant correlation among the expression levels of BRCA1 ,ERCC1 mRNA and clinical efficacy (P<0.01). (3)ATP-TCA and detection of drug resistance genes combined to predict the clinical application of PTX + CBP resistance may occur in 8/9 cases. Conclusions ATP-TCA may be an ideal method of in vitro drug sensitivity testing method, which could effectively predict clinical chemotherapy sensitivity. Combination of the drug-resistant associated genes detection method and the ATP-TCA method can increase the predictive effectiveness of ovarian cancer chemosensitivity and guide individual chemotherapy of ovarian cancer.     

子宫畸形合并妊娠母儿妊娠结局临床分析

目的探讨妊娠合并子宫畸形对于母儿妊娠结局的影响。方法选取北京大学人民医院收治的妊娠合并子宫畸形患者108例,并以372例正常子宫妊娠患者作为对照,对其临床资料进行回顾性分析。结果①在29245例分娩的病例中,妊娠合并子宫畸形共108例,发生率约为0.4%。其中以子宫纵膈最为常见,占49.1%,其次为双子宫,占21.3%;②子宫畸形合并妊娠的患者中,胎位异常发生率（46.3%）及剖宫产率（73.2%）均显著升高;③子宫畸形组的平均孕周（37.2周）,新生儿平均出生体重（2873g）显著低于对照组,而早产率（24.1%）及足月低体重儿发生率（6.5%）均显著高于对照组;④纵膈子宫、单角子宫（83.3%合并残角子宫）、双角子宫、及双子宫在终止妊娠方式上存在差异,剖宫产率在纵膈子宫为最低（64.2%）。各组在早产率、孕周、胎儿体重方面差异无统计学意义（P〉0.05）。结论子宫畸形对于妊娠结局可产生不良影响,临床医生应加强孕前及孕期管理。     

"Primary peritoneal" high-grade serous carcinoma is very likely metastatic from serous tubal intraepithelial carcinoma: assessing the new paradigm of ovarian and pelvic serous carcinogenesis and its implications for screening for ovarian cancer

Objective

Primary peritoneal high-grade serous carcinoma is thought to arise from the peritoneum, but recent data suggest that the fallopian tube may be an occult source of many of these tumors. This study was performed to evaluate this hypothesis in an unselected series of cases.

Methods

Fallopian tubes from 51 consecutive cases meeting the GOG criteria for primary peritoneal high-grade serous carcinoma, FIGO stages II-IV, were analyzed.

Results

Serous tubal intraepithelial carcinoma (STIC) was identified in 19 patients (37%). When the fimbriae were examined, STIC was identified in 46%, and when all tubal tissue was examined, 56%. STIC was confined to the fimbriae in 53%, involved fimbriae and nonfimbrial mucosa in 20%, and was confined to nonfimbrial mucosa in 20%. Patients with STIC were significantly older than those without STIC (75 years vs. 67 years, respectively; p = 0.007). Patients with STIC were significantly more likely to have FIGO stage IV disease as compared to those without STIC (42% vs. 12.5%, respectively; p = 0.037).

Conclusions

At least half the cases of primary peritoneal high-grade serous carcinoma are associated with intraepithelial carcinoma of the fallopian tube, usually involving the fimbriae. These findings support the view that, like “primary ovarian carcinoma,” what has been traditionally classified as “primary peritoneal carcinoma” is probably derived from occult high-grade serous carcinoma in the fallopian tube. These findings have important implications for ultrasound screening trials for ovarian cancer which are based on the assumption that an enlarged ovary is a very early manifestation of disease.     

CCL11‐induced eosinophils inhibit the formation of blood vessels and cause tumor necrosis

We previously demonstrated that IL‐18 and CCL11 were highly expressed in an NFSA tumor cell line that showed limited angiogenesis and severe necrosis. However, IL‐18 was not responsible for the immune cell accumulation and necrosis. Here, we attempted to clarify the relevance of CCL11 in angiogenesis and tumor formation. We established CCL11‐overexpressing MS‐K cell clones (MS‐K‐CCL11) to assess the role of CCL11 in immune cell accumulation and angiogenesis. The MS‐K‐CCL11 cells did not form tumors in mice. MS‐K‐CCL11‐conditioned medium (CM) and recombinant CCL11 induced macrophage and eosinophil differentiation from bone marrow cells. The MS‐K‐CCL11‐CM effectively recruited the differentiated eosinophils. Furthermore, the eosinophils damaged the MS‐K, NFSA and endothelial cells in a dose‐dependent manner. Administration of an antagonist of CCR3, a CCL11 receptor, to NFSA tumor‐bearing mice restored the blood vessel formation and blocked the eosinophil infiltration into the NFSA tumors. Furthermore, other CCL11‐overexpressing LM8 clones were established, and their tumor formation ability was reduced compared to the parental LM8 cells, accompanied by increased eosinophil infiltration, blockade of angiogenesis and necrosis. These results indicate that CCL11 was responsible for the limited angiogenesis and necrosis by inducing and attracting eosinophils in the tumors.