制备脱细胞真皮基质的实验研究

目的 采用高渗盐一胰蛋白酶法制备脱细胞真皮基质(Acellular dermal matrix,ADM)并进行组织学观察和微生物学检测,探讨这种方法的可行性和机制。方法 SD大鼠4只,分别麻醉后以8％硫化钠溶液脱去躯干部皮毛,常规消毒铺巾,切取大鼠背部全层皮肤制成厚约0.4mm的中厚皮,1mol／LNaCl溶液37℃浸泡48h分离表真皮,以0．25％胰蛋白酶 0.1％乙二酸二乙胺(EDTA)溶液37℃分别热消化60、70、80、90min脱去细胞成分,并进行冷冻干燥处理。ADM作光镜、电镜观察和微生物学检测并比较分析每只大鼠不同时间消化处理的ADM的差别和不同大鼠经相同处理得到的ADM的差别。结果消化时间不同的ADM均具有柔软、一定的柔韧性和伸展性、弹性好、弯曲不断裂的特点,组织学观察见表皮细胞均完全去除,但是消化90min,ADM的基底膜(Basement membrane complex,BMC)和胶原纤维的结构与排列有不同程度的破坏;消化60、70min的ADM虽然基底膜和胶原纤维的结构与排列正常存在,但脱细胞不完全;消化80min制备的ADM组织学检查基底膜完整连续．胶原纤维、弹力纤维结构及排列完整规则,没有细胞成分存在。不同SD大鼠经同样的处理所得的ADM一般形状和组织学没有明显差别。微生物学检测没有细菌生长。结论采用高渗盐一胰蛋白酶法制备脱细胞真皮基质的方法能达到既完全脱去细胞成分又使基底膜完整连续,胶原纤维和弹力纤维结构及排列正常的双重目标,ADM柔韧性好、不断裂,无细菌生长。     

浅表食管癌侵袭深度及分化程度与区域淋巴结转移的临床分析

目的探讨浅表食管癌侵袭深度及分化程度与区域淋巴结转移的关系。方法本院2002年6月至2004年6月总共手术治疗食管癌923例,其中早期浅表食管癌68例,本文就对其术后侵袭深度及分化程度与区域淋巴结转移情况进行回顾性分析。结果本组无手术死亡,全组淋巴结转移率27%,其中高出黏膜型食管癌的淋巴结转移率为38%,侵袭越深,淋巴结转移率越高;分化程度越低淋巴结转移率越高。结论高度重视浅表型食管癌的根治性,对早期食管癌也应按肿瘤外科的原则行食管的次全切除术并常规清扫区域淋巴结。     

Prognostic significance of resident CD103+CD8+T cells in human colorectal cancer tissues

The prognostic significance and clinical implications of resident CD103⁺CD8⁺T cells in human colorectal cancer tissues still remains largely unexplored. In our present study, we aimed to characterize the resident CD8⁺T cells in human colorectal cancer tissues by using double staining of CD103 and CD8, and further evaluated the prognostic significance of resident CD8⁺T cells in colorectal cancer. We found that the OS rate of the colorectal cancer patients with higher infiltration of CD8⁺T cells, or with higher numbers of resident CD103⁺CD8⁺T cells, or with higher ratio of CD103⁺CD8⁺T cells over total CD8⁺T cells in cancer tissues was significantly better than that of the patients with lower infiltration of CD8⁺T cells, or with lower numbers of resident CD103⁺CD8⁺T cells, or with higher ratio of CD103⁺CD8⁺T cells over total CD8⁺T cells in cancer tissues, respectively. Moreover, higher infiltration of CD8⁺T cells in colorectal cancer tissues was significantly and inversely correlated with advanced TNM stage. Higher numbers of resident CD103⁺CD8⁺T cells in colorectal cancer tissues were significantly and inversely correlated with distant metastasis status. Higher ratio of CD103⁺CD8⁺T cells over total CD8⁺T cells in colorectal cancer tissues was significantly and inversely correlated with age status. The COX model analysis demonstrated that higher infiltration of CD8⁺T cells, higher numbers of resident CD103⁺CD8⁺T cells, or higher ratio of CD103⁺CD8⁺T cells over total CD8⁺T cells in colorectal cancer tissues, could serve as independent prognostic predictors for colorectal cancer patients. Taken together, our present study demonstrated the density of tumor infiltrating CD8⁺T cells or the numbers of resident CD103⁺CD8⁺T cells in colorectal tissues could be used as an important prognostic predictor for this malignancy.     

Chloride channel 3 (CIC-3) predicts the tumor size in hepatocarcinoma

Chloride channel 3 (CIC-3) has been suggested to be implicated in the carcinogenesis though; it still remains ill understood in hepatocarcinoma, especially in terms of clinicopathological meaning of its expression. Given this, herein, to understand the clinicopathological significance of CIC-3 expression in hepatocarcinoma, Immunohistochemistry was performed to examine the level of CIC-3, followed by statistical analysis of the correlation between expression versus clinicopathological variables, including gender, age, TNM classifications, tumor size, lymph node metastasis and overall prognosis. It was shown that positive staining of CIC-3 can be present in both hepatocarcinoma and its paired normal controls; and that CIC-3 was significantly over-expressed in hepatcarcioma on the whole relative to paired normal controls. Moreover, up-regulation of CIC-3 markedly correlated with tumor size and overall prognosis, suggesting that CIC-3 expression could predict both tumor size and overall prognosis in hepatocarcinoma.     

The clinical significance of plasma CFHR 1–5 in lupus nephropathy

A deficiency of complement factor H may lead to excessive consumption of C3 and an increase in C3b deposition, which are important pathological characteristics of lupus nephritis. Complement factor H-related proteins (CFHRs), comprising CFHR1 to CFHR5 (CFHR1–5), are members of the wider factor H/CFHR family. Their role in lupus nephritis remains unclear. In this study, we compared circulating levels of CFHR1–5 in 152 patients diagnosed with lupus nephritis and 20 unrelated healthy individuals to explore the relationship between the expression of CFHR1–5 and development of the disease. We found that plasma levels of CFHR3 and CFHR5 were higher in patients with lupus nephritis than in healthy individuals; also, CFHR3 and CFHR5 concentrations increased with increasing systemic lupus erythematosus disease activity index (SLEDAI) values (P < 0.05). Pearson's and Spearman's correlation test results confirmed that plasma CFHR3 and CFHR5 levels in lupus nephritis patients were positively correlated with proteinuria and levels of creatinine (Cr) and anti-dsDNA (correlation coefficients = 0.491–0.717, P < 0.05), while they were negatively correlated with plasma C3 levels and eGFR [correlation coefficients = –(0.706–0.788), P < 0.05]. Receiver operating characteristic (ROC) curve analysis results confirmed that plasma CFHR3 and CFHR5 levels were predictive of SLEDAI values and disease end points (area under the curve = 0.664–0.884, P < 0.05), with patients with both high CFHR3 and high CFHR5 exhibiting the shortest progression-free survival. Thus, both CFHR3 and CFHR5 are of prognostic value in lupus nephritis status.     

High genetic diversity and recombination events of porcine astrovirus strains identified from ill and asymptomatic pigs in 2017, Hunan Province,China

Astroviruses (AstV) are associated with enteric and systemic disease in mammals and birds. Astroviruses have received increased attention recently as they have been found to be associated with sporadic neurologic disease in mammals including humans. In pigs, porcine astrovirus (PoAstV) can be widely detected and has been grouped in five genotypes (PoAstV1 to PoAstV5). In the present study, we detected multiple PoAstVs in serum samples, nasal swabs, and fecal swabs collected from pigs suffering from respiratory disease or diarrhea but also from asymptomatic pigs, indicating a wide tissue tropism of the identified PoAstV genotypes. Coinfection of different genotypes in the same pig was commonly observed, and within an individual pig a high genetic diversity was observed for viruses belonging to the same PoAstV genotype. Two complete genomes of PoAstV2-WG-R2/2017 and PoAstV4-WG-R2/2017 were successfully obtained and characterized, with genome sizes of 6396 and 6643 nucleotides, respectively. The PoAstV2-WG-R2/2017 genome showed identities of 67.2–77.4% to other known PoAstV2 genomes, and the PoAstV4-WG-R2/2017 genome showed identities of 72.8–80.5% to other known PoAstV4 genomes. The predicted spike domain of open reading frame 2 (ORF2) of these strains showed the highest genetic heterogeneity, with amino acid identities of 13.7–70.9% for PoAstV2-WG-R2/2017 to other known PoAstV2 strains, and identities of 24.4–63.3% for the PoAstV4-WG-R2/2017 to other known PoAstV4 strains. Possible recombination events were identified in each of the two sequences. Two subclades of PoAstV2 and three subclades of PoAstV4 were defined in the present analyses. The obtained data provide further evidence for extraintestinal infectivity of PoAstVs, and confirmed the high genetic diversity of PoAstVs and the coinfection potential of different PoAstV types in a single pig.

     

Further confirmation of second- and third-generation Eimeria necatrix merozoite DEGs using suppression subtractive hybridization

Parasitology Research - In our previous study, we obtained a large number of differentially expressed genes (DEGs) between second-generation merozoites (MZ-2) and third-generation merozoites (MZ-3)...     

Potential application of shape memory plastic as elastic material in clinical orthodontics

Polynorbornen, a shape memory plastic developed in Japan, has a glass transitional point of 35 degrees C. Once the environmental temperature exceeds the critical point, this plastic will begin to display an elastic property, then return to its original shape, if deformed. We examined whether the force generated during the elastic phase of polynorbornen could be used to displace human teeth. We found that the shape memory plastic wire of 1 mm in diameter stretched to two to three times of its original length at a temperature of 50 degrees C and a speed of 0.5 mm/sec would exert a relatively stable continuous light force of 119-156 g to move the teeth. This new material, compared with conventional elastic modules used in orthodontic therapy, exhibited a lesser degree of force degradation at a body temperature of 37 degrees C for a long period, and can be manufactured to near the tooth colour required. These advantages make feasible clinical application of the shape memory plastic in orthodontics.     

Processing of ameloblastin by MMP-20

Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro(2), Gln(130), Gln(139), Arg(170), and Ala(222). This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr(223), as well as the 17-kDa (Val(1)-Arg(170)) and 15-kDa (Val(1)-Gln(130)) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.     

Mutational analysis of candidate genes in 24 amelogenesis imperfecta families

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited defects in dental enamel formation. The malformed enamel can be unusually thin, soft, rough and stained. The strict definition of AI includes only those cases where enamel defects occur in the absence of other symptoms. Currently, there are seven candidate genes for AI: amelogenin, enamelin, ameloblastin, tuftelin, distal-less homeobox 3, enamelysin, and kallikrein 4. To identify sequence variations in AI candidate genes in patients with isolated enamel defects, and to deduce the likely effect of each sequence variation on protein expression and structure, families with isolated enamel defects were recruited. The coding exons and nearby intron sequences were amplified for each of the AI candidate genes by using genomic DNA from the proband as template. The amplification products for the proband were sequenced. Then, other family members were tested to determine their genotype with respect to each sequence variation. All subjects received an oral examination, and intraoral photographs and dental radiographs were obtained. Out of 24 families with isolated enamel defects, only six disease-causing mutations were identified in the AI candidate genes. This finding suggests that many additional genes potentially contribute to the etiology of AI.