胚胎胰组织移植的实验研究

目的：研究胚胎胰组织在同种异体内再生长发育的可能性，并根据不同胎龄、不同移植部位的移植胚胰生长状况，研究大鼠胚胰移植的适合胎龄和移植部位。方法：将远交系SD大鼠妊娠15．5d（E15．5）、E16．5、E17．5和E18．5的胚胰移植到成年健康SD大鼠的肾包膜下或网膜内。移植后14～21d期间剖腹观察器官生长情况，并取标本行病理切片和免疫组化观察。结果：①E15，5胚胰植入肾包膜21d后，发育良好，腺泡导管分化发育，β细胞增殖并聚集。植入网膜内16d后，有中度排斥反应，发育有限。②E16．5胚胰植入肾包膜下21d后，可见中度排斥，但结构发育较好。③E17．5和E18．5胚胰植入肾包膜下后，16d内就发生重度排斥，生长严重受限，β细胞也未见增殖。④E15．5胚胰移植到小鼠体内，未经任何免疫抑制处理，16d内见到重度排斥表现并导致组织坏死。结论：不使用免疫抑制剂时，一定胎龄的胚胰可在同种远交系成年大鼠体内再血管化，并发育为近似正常胰腺组织。E15．5或更早期的胚胰用来移植比较适合，排斥反应较轻，增殖潜力较大。胚胰移植在肾包膜下相对于移植在网膜内的排斥轻、发育好。

胰岛移植治疗胰岛素依赖型糖尿病

14例Ⅰ型糖尿病,病程最短1年,最长11年。胰岛素用量每日22～60单位,其中4例反复发作酮症酸中毒。14例患者均系病情控制后,应用人胎胰岛行大网膜囊腔内移植,并随访6～20个月,发现12例有效,胰岛素每日用量减少30％以上。10例在移植10～14天时,有明显低血糖反应。研究表明,冷冻复苏胰岛细胞和未经冷冻胰岛细胞功能相似,光镜下两者组织细胞均生长良好。

MRI对胰腺囊性肿瘤的诊断价值

目的探讨MRI对胰腺囊性肿瘤的诊断价值。方法回顾分析23例经手术病理证实的胰腺囊性肿瘤资料,总结其影像表现。结果黏液性囊性肿瘤10例,囊性肿块较大,肿块平均直径超过10 cm,由较厚的纤维壁分隔成多房囊肿,外壁光滑,边界清楚。浆液性囊腺瘤6例,病灶较小,囊内有分隔而呈放射状排列,中心瘢痕为其特征性表现。胰腺囊性转移瘤3例,均有肿瘤病史,表现不典型,需结合肿瘤病史定性。胰胚细胞瘤2例,幼儿发病,肿块巨大,肿瘤坏死囊变,形成假-假性囊肿。胰腺乳头状囊实性肿瘤1例,表现为边界清楚的囊实性肿块,囊壁上有壁结节。胰腺毛细血管瘤1例,表现与浆液性囊腺瘤相似,难以术前诊断。结论MRI对胰腺囊性肿瘤具有高度敏感性,不同病变有一定的特征性表现, 对术前定性极有价值。

胰母细胞瘤1例报告

胰母细胞瘤系极为罕见的儿童胰腺低度恶性肿瘤。我院于1987年12月遇到1例,现报告如下。患者男,8岁。上腹部疼痛1年,腹部肿物1个月,无其他不适,于1987年12月入院。查体:腹平坦,上腹部可扪及约9×6cm肿物,质硬,表面光滑,活动度差。肝功正常,血清淀粉酶8u,硷性磷酸酶146.

Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

AIM： To enhance the differentiation of insulin producing cell （IPC） ability from embryonic stem （ES） cells in vitro. METHODS： Four-day embryoid body （EB）-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator （Amaxa biosystems, Germany） in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR （SQ-PCR） detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixll, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry （IHC）, and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease （SCID） pretreated with streptozotocin （STZ） were used to eliminate plasma glucose restoration after pax4^＋ ES implantation. RESULTS： A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixll, pdxl, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION： Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasrnid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, the potential of pax-4-nucleofected cells in medical treatment is promising.

Wnt/β-catenin signaling pathway is active in pancreatic development of rat embryo

AIM： To elucidate the role of Wnt/β-catenin signaling pathway in pancreatic development of rat embryo. METHODS： The mRNAs of β-catenin, APC, cyclin D1 genes were amplified by means of semiquantitative reverse transcription polymerase chain reaction （RTPCR） from embryonic pancreas in different periods and normal pancreas of rat, respectively. Protein expression of these genes in embryonic pancreas of E14.5-E18.5 was examined by immunohistochemical method. RESULTS： In embryonic pancreas of E14.5, the transcript amplification of β-catenin and cyclinD1 genes was detected. In embryonic pancreas of E18.5, the transcription levels of β-catenin and cyclinD1 genes became much higher than in other periods. But in adult rat pancreas the transcription of cyclinD1 gene could not be observed. Only until E18.5, the transcript amplification of mRNA of APC gene could be detected. Surprisingly, the transcription level of APC gene became much higher in adult rat pancreas than in embryonic pancreas. By means of immunohistochemical staining, identical results were obtained to the above by RP-PCR, except for β-catenin protein in adult rat pancreas. CONCLUSION： Active Wnt/β-catenin signaling occurs in rat embryonic pancreas and is probably important for pancreatic development and organ formation.

The oncofetal protein IMP3 is an indicator of early recurrence and poor outcome in mucoepidermoid carcinoma of salivary glands

Objective: Mucoepidermoid carcinoma(MEC) is the most common primary malignancy of the salivary glands. Insulin-like growth factor-II m RNA-binding protein-3(IMP3) is an important prognostic factor in some cancers and a tool that differentiates between benign and malignant pancreatic lesions. This study aimed to identify a relationship between the expression of IMP3 and the outcome of salivary gland MEC, as well as to differentiate MEC from pleomorphic adenoma(PA).Methods: Tissue specimens from 70 cases of salivary gland MEC, 40 cases of PA, and 10 cases with normal salivary gland were examined immunohistochemically for IMP3. The association among the expression of IMP3, clinicopathological characteristics and patient's survival was assessed.Results: IMP3 was present in 51.4% of MEC but absent in PA and normal salivary gland tissues. IMP3 expression was associated with age > 60 years, submandibular gland tumors, tumor size > 4 cm, high-grade tumors, lymph node metastasis, involvement of surgical margins, perineural invasion, distant metastasis, advanced TNM stage, tumor relapse, and death(P<0.05). Increased expression of IMP3, tumors of the submandibular gland, and lymph node metastasis were independent prognostic factors of disease-free survival(DFS). In addition, IMP3 was a strong predictor of overall survival(OS) together with distant metastasis and intermediate and high-grade tumors.Conclusions: IMP3 expression is highly important in evaluating the outcome of MEC. IMP3 can be used to differentiate MEC from PA of salivary glands.

In vitro derivation of functional insulin-producing cells from human embryonic stem cells

The capacity for self-renewal and differentiation of human embryonic stem （ES） cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid （RA） was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdxl and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin （STZ）-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdxl, glucokinase, nkx6.1, lAPP, pax6 and Tcfl. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.METHODS： Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body （EB） culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone （DTZ）, a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells.The outgrowths were incubated in DTZ solution （final concentration, 100μg/mL） for 15 rain before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA.RESULTS： DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells （Nkx-ES cells）, which was as much as 2 wk earlier, than those in the culture of parental ES cells （wt-ES）. The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 （PDX1）, proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters.Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.CONCLUSION： Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting 13 cells from pluripotent mouse embryonic stem cells （ESCs）.The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of 13 cells such as insulinl, insulin2, Isletl, MafA, insulinoma-associated antigen 1 （IA1） and so on, indicating a similar gene expression pattern to adult islet 13 cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement 13 cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

Utility of endoscopic ultrasound, cytology and fluid carcinoembryonic antigen and CA 19-9 levels in pancreatic cystic lesions

AIM: To assess the diagnostic accuracy of endoscopic ultrasound (EUS), fluid tumor markers and cytology in distinguishing benign from (pre)malignant pancreatic cystic lesions. METHODS: 46 consecutive patients, referred to a gastroenterologist and surgeon for a symptomatic or incidental pancreatic cyst, were reviewed. EUS, cytology, and carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) levels were compared with the final diagnosis, based on surgical pathology and/or imaging follow-up of at least 12 mo. Cysts were classified as benign (pseudocyst, serous cystadenoma) or malignant/ pre-malignant (mucinous cystic neoplasm). Receiver- operator characteristics (ROC) curve analysis was performed. RESULTS: The mean age was 56 years; 29% were male and median cyst diameter was 3 cm. Final outcome was obtained in 41 (89%) patients. Twenty-three (56%) of these 41 had surgical pathology. Twenty-three (56%) had benign lesions and 18 (44%) had malignant/pre- malignant lesions. Sensitivity, specificity and positive and negative predictive value of EUS alone to distinguish benign from malignant/premalignant pancreatic cystic lesions were 50%, 56%, 36% and 54% and for cytology were 71%, 96%, 92% and 85%, respectively. The corresponding values for the ROC-derived ideal cutoffswere 75%, 90%, 75%, 90% for CA 19-9 (> 37 U/mL) and 70%, 85%, 79% and 78% for CEA (> 3.1 ng/mL). Subgroup analysis of those with surgical pathology yielded almost identical performance and cutoffs. CONCLUSION: Cytology and cyst fluid tumor marker analysis is a very useful tool in distinguishing benign from (pre)malignant pancreatic cystic lesions.

Culture and Identification of Myoblasts Isolated from Duck Embryos

Using embryonic myoblasts to research the formation and de-velopmental mechanisms of skeletal muscle is becoming a research hotspot. This study aimed to establish a method of isolation, culture and identification of my-oblasts in duck embryos. [Method] Pectoral and leg muscle samples were isolated from the embryos of Gaoyou duck at 13 d of hatching, then disassociated with col-lagenase and trypsin and purified via differential adhesion. The isolated cells were cultured in vitro and detected for the expression of Pax7 protein using immunofluo-rescence technique. [Result] Myoblasts were obtained successful y both from pectoral and leg muscles in duck embryos and these cells proliferated strongly and differen-tiated wel . Immunofluorescence staining showed that more than 95% cells could express Pax7 protein. [Conclusion] In summary, we report the successful establish-ment of a complete system for the isolation, purification, identification and culture of myoblasts from duck embryos.

Development of a Synthetic Medium for the in Vitro Culture of Bovine Embryos

The aim of this study was to develop a synthetic medium for the in vitro culture of bovine embryos, using various growth factors and cytokines （GF-CYK）： insulin-like growth factorl （IGF-Ⅰ）, insulin-like growth factorⅡ （IGF-Ⅱ）, basic fibroblast growth factor （bFGF）, leukemia inhibitory factor （LIF）, granulocyte-macrophage colony stimulating factor （GM-CSF） and transforming growth factor beta Ⅰ （TGF-β1） ＋ hyaluronan （HA） ＋ recombinant albumin （RA）. The embryos were cultured in synthetic oviduct fluid （SOF） supplemented with： treatment 1 （T1）： bovine serum albumin （BSA） ＋ insulin, transferrin and selenium （ITS） （control）; or treatment 2 （T2）： GF-CYK ＋ HA ＋ RA. The blastocyst rates were not significantly different between TI and T2, at seven days post fertilization （dpf） （28.9% ± 2.4% and 31.8% ±2.2%）, and at 8 dpf （36.5% ±2.4% and 39.1% ±1.9%）, respectively （P 〉 0.05）. The total cell number （TCN） was significantly higher with T2 than that with T1 at 7 dpf（164.9 ±5.3 and 149.7 ±4.0） and 8 dpf （182.7 ±6.4 and 165.0 ±5.5） （P 〈 0.05）. The blastocyst diameter obtained with T2 was significantly greater （P 〈 0.05） than with T1 at 7 dpf （173.3 μm ±4.9 μm and 157.2μm ±4.1 μm, respectively）, however, no significant differences were observed at 8 dpf （190.3 μm 5.2 μm and 179.7 μm ± 5.3 μm, respectively）. In conclusion, the synthetic medium （T2） shows a comparable development rate to the control medium and improves the blastocyst diameter and the TCN.

Cell therapy in diabetes: current progress and future prospects

Diabetes mellitus, characterized by the impaired metabolism of insulin secretion in β cells, is becoming one of the most prevalent diseases around the world. Recently, cell replacement based on differentiation of various pluripotent stem cells, including embryonic stern cells, induced pluripo- tent stem cells and multipotent stem cells, such as bone mar- row mesenchymal stem cells, adipose-derived stem cells and gnotobiotic porcine skin-derived stem cells, is becoming a promising therapeutic strategy. Cells derived from pancreatic tissues or other tissues that are relevant to β cell differentiation have also been used as cell source. However, in spite of hopeful experimental results, cell therapy in diabetes still confronts certain obstacles, such as purity of cells, functional differentiation of stem cells and possible tumorigenesis, which, in turn, lead to the seeking of new-generation tools, such as xenogenetic materials. In this review, we will sum- marize the current knowledge and future prospects of cell therapy in diabetes mellitus.