Laser-induced fluorescence spectroscopy for in vivo diagnosis of non-melanoma skin cancers

BACKGROUND AND OBJECTIVES: Laser-induced fluorescence spectroscopy is a non-invasive technique previously used for detection of cancer in a variety of organ systems. The objective of this study was to determine whether in vivo laser-induced fluorescence spectroscopy alone at the visible excitation wavelength of 410 nm could be used to detect non-melanoma skin cancers. STUDY DESIGN/MATERIALS AND METHODS: The system consisted of a nitrogen/dye laser tuned at 410 nm, an optical multichannel analyzer, and a fiber optic probe for excitation of tissue and collection of fluorescence emission. Two hundred and seventy nine measurements were performed from normal and abnormal tissues in 49 patients. Patients were classified as having either skin types I, II, or III. Biopsy of the abnormal tissues were then performed. Each measurement was assigned as either normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), pre-cancerous, or benign. Total emission photon count was used as the discriminating index. A threshold value was calculated to separate normal tissue indices from indices of cancer tissues. The classification accuracy of each data point was determined using the threshold value. RESULTS: Cancers were classified 93, 89, and 78% correctly in patients with skin types I, II, and III, respectively. Normal tissues were classified 93, 88, and 50% correctly in patients with skin types I, II, and III, respectively. Using the same threshold, pre-cancerous spectra were classified 78 and 100% correctly in skin types I and III, respectively. Benign lesions were classified 100, 46, and 27% correctly in patient with skin types I, II, and III, respectively. CONCLUSIONS: In vivo laser induced fluorescence spectroscopy at 410 nm excitation and using the intensity of emission signal is effective for detection of BCC, SCC, and actinic keratosis, specially in patients with light colored skin.     

表达增强型绿色荧光蛋白的骨肉瘤细胞亚株建立及生物学特性

目的 建立经绿色荧光蛋白基因转染的骨肉瘤细胞亚株并研究其生物特性。方法 利用脂质体转染增强型绿色荧光蛋白真核表达质粒（pEGFP-N1）人人骨肉瘤MG63细胞系，通过有限稀释法和细胞电泳，获得两株细胞克隆M6和M8，经体外细胞增殖、软琼脂形成、生长曲线、裸鼠成瘤试验综合分析其生物学行为改变。应用组织形态学观察、染色体分析、软琼脂克隆形成法研究癌细胞的生物学特性。结果 M6和M8两株在细胞电泳率和侵袭性上差异有统计学意义（P〈0．05），其中M6的群体倍增时间为38．4h，软琼脂形成率为18．7％，M8群体倍增时间为23．0h，软琼脂形成率为29．3％。裸小鼠背部皮下接种M6和M8，发现M8成瘤时间短，细胞增殖快，但在4周内两者均不发生转移。结论 骨肉瘤细胞亚系有不同的转移特性，GFP的整合及表达未对MG63细胞的生长状态造成明显影响，可作为报告基因进一步了解骨肉瘤细胞转移的差异性分析。     

骨髓干细胞参与小鼠急性肾小管坏死修复的实验研究

【摘要】 目的 观察在生理和病理情况下,骨髓来源的干细胞(BMSC)能否分化成肾小管上皮细胞。 方法 绿色荧光蛋白(GFP)标记的C57BL/6转基因小鼠提供骨髓细胞。同种无荧光标记的C57BL/6小鼠100只分为正常对照组、全身照射组、缺血再灌注组、骨髓移植组、骨髓移植+缺血再灌注组。受体鼠的骨髓重建经血液常规检查和流式细胞仪检测确认,并采用荧光组织化学、免疫组织化学等方法观察绿色荧光标记的BMSC在受体鼠肾脏的分布及数量。 结果 全身致死剂量γ射线照射未造成小鼠肾脏组织结构和生理功能的明显改变。骨髓移植后第56、84天的受体鼠肾小管中有少量GFP阳性细胞的存在[(78.75±5.99)%、(79.58±4.60)%],激光共聚焦显微镜进一步证实这些细胞位于肾小管,并表达肾小管上皮细胞特异性的功能蛋白megalin。 结论 在生理和病理情况下,骨髓干细胞均可以向肾小管上皮细胞转分化,参与肾小管上皮细胞的更新,并且在急性肾小管坏死的病理条件下,骨髓干细胞的肾向转化率与肾脏受损程度有关。     

Characterization of EGFP-labeled mesenchymal stem cells and redistribution of allogeneic cells after subcutaneous implantation

INTRODUCTION: Bone marrow mesenchymal stem cells (MSCs) are ideal target cells for cell transplantation and tissue engineering. We investigated their biological characteristics and differentiation mediated by different methods. It is important to study the short-term fate of labeled allogeneic MSCs after subcutaneous implantation in rabbits in order to provide insights into the application of allogeneic MSCs for tissue regeneration. MATERIALS AND METHODS: Mesenchymal stem cells were labeled by two different methods in vitro and then were incubated with gelatin sponge. Autologous MSCs-Gelatin constructs and allogeneic MSCs-Gelatin constructs were subcutaneously implanted into 32 rabbits. The constructs were analyzed for the survival and migration of labeled MSCs at day 3, week 1, 3, and 5 post-implantation. RESULTS: EGFP was successfully expressed following transfection of MSCs with the retroviral vector pLEGFP-N1. In addition, EGFP-MSCs can be functionally induced into osteocytes, chondrocytes, and adipocytes in conditioned media. By weeks 3 after implantation, the labeled cells distributed extensively on the surface of gelatin sponge and gradually integrated into host tissues. EGFP-labeled MSCs were observed under fluorescence microscopy and BrdU-expressing cells were detected with immunohistochemical stain in allogeneic or autologous MSCs-Gelatin constructs during the initial five weeks after implantation. CONCLUSIONS: It is a simple and reliable way to trace the changes of MSCs in vivo by EGFP in cell transplantation and gene therapy. Allogeneic rabbit MSCs can survive for at least 5 weeks after subcutaneous implantation and maintain a strong ability of migration, indicating that allogeneic MSCs are of certain value in clinical application for temporary replacement.     

移植骨髓间充质干细胞对兔退变椎间盘髓核细胞凋亡的影响

目的 观察骨髓间充质干细胞(MSCs)移植对兔退变椎间盘髓核细胞凋亡的影响.方法 以各兔L2/3、L3/4、L4/5、L5/6节段分为正常组、退变组、成纤维细胞(SFs)移植对照组、MSCs移植治疗组.MSCs和SFs分别经绿色荧光蛋白(GFP)转染后,注射植入退变椎间盘的髓核.通过透射电镜观察退变椎间盘凋亡髓核细胞形态;用实时定量聚合酶链反应(PCR)检测退变组织中髓核细胞凋亡相关基因bcl-2和box mRNA的表达;免疫荧光法标记髓核细胞凋亡相关蛋白Caspase-3,并通过TUNEL法标记凋亡髓核细胞,激光共聚焦显微镜检测髓核细胞凋亡蛋白表达率和细胞凋亡比率.结果 透射电镜下,退变椎间盘中凋亡髓核细胞呈现出核染色质边集,空泡形成,核膜断裂,凋亡小体形成等变化.MSCs移植治疗组bcl-2 mRNA的表达量高于退变组和SFs移植对照组(P<0.05),bax mRNA的表达量与退变组差异无统计学意义(P>0.05).MSCs移植治疗组细胞凋亡率和Caspase-3表达率均高于正常组[细胞凋亡率分别为(16.75±2.14)%和(6.86±1.08)%;Caspase-3表达率分别为[(20.34±1.03)%和(6.09±0.77)%](P<0.05),低于退变组和SFs移植对照组[细胞凋亡率分别为(31.87±4.16)%和(29.02±2.16)%;Caspase-3表达率分别为(31.50±3.78)%和(30.20±4.93)%](P<0.05).结论 髓核细胞凋亡在椎间盘退变过程中起重要作用.MSCs移植能有效抑制椎间盘髓核细胞凋亡,延缓椎间盘退变过程.     

与基底细胞癌细胞凋亡相关的分子生物学研究--促凋亡分子Bad的克隆及其pEGFP-C3真核表达载体的构建

目的:克隆Bcl-2相关凋亡基因Bad并构建其真核表达载体,探讨其对肿瘤细胞凋亡的诱导作用。方法:采用RT-PCR的方法,扩增Bad基因全长片段。通过基因定向克隆,构建Bad基因的真核表达质粒载体。结果:经酶切鉴定、PCR及DNA序列测定鉴定,Bad基因表达质粒pEGFP-C3载体成功构建。结论:成功克隆Bcl-2相关促凋亡基因Bad并构建其真核表达载体pEGFP-C3-Bad,为进一步研究Bad在人肿瘤细胞系中的促凋亡作用奠定了实验基础。     

携带报告基因EGFP的CXCR4 RNA干扰质粒的构建及生物活性的鉴定

目的构建携带报告基因EGFP的CXCR4RNA干扰质粒,证实其高效抑制靶基因表达的生物活性。方法设计有发夹状结构的两条DNA序列,用PCR扩增CXCR4特异性小干扰RNA(smallinterferingRNA,siRNA),转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的pGensil-1质粒,转化DH5a菌株,提取质粒行酶切及测序鉴定。重组质粒转染PC-3m细胞株,检测绿色荧光蛋白表达及对细胞CXCR4表达的抑制情况。结果成功构建了CXCR4靶向的RNA干扰质粒pGensil-1/siCXCR4,在靶细胞可同时表达siRNA及绿色荧光蛋白。结论pGensil-1/siCXCR4质粒的成功构建对利用基因干扰技术治疗前列腺癌转移方面的研究奠定了一定的实验基础。     

Blue dye single labelling for colorimetric sentinel lymph node mapping in early endometrial cancer: A feasibility study

BackgroundSurgical staging in endometrial cancer includes a systematic lymphadenectomy with significant morbidity, although its therapeutic role is unclear. Sentinel lymph node (SLN) study is a less morbid alternative to identify nodes most likely to be metastatic, permitting selective removal and thus reducing morbidity without compromising oncological safety. This study was done using blue dye single labelling to study the feasibility and utility in identifying SLN in early disease.MethodsTwenty-two patients of early-stage low-risk disease during surgical staging underwent cervical injection of methylene blue, SLN mapping, and sampling as per the standard algorithm, followed by a systematic lymphadenectomy in all cases. SLN were submitted separately for ultrastaging (US).ResultsTwenty patients underwent the procedure, and SLN could be identified in 18 patients with an overall mapping rate of 90% with a bilateral mapping rate of 70%, and a negative mapping rate of 10%. 57 SLN were identified along with two suspicious non-sentinel nodes and 11 were metastatic on US with a sensitivity of 66.7% and NPV of 87.5%. All patients with metastatic nodes, however, could be identified by applying the standard SLN algorithm for sampling.ConclusionSLN mapping algorithm with blue dye single labelling in early endometrial cancer, by identifying LN most likely to be metastatic enabling their selective removal may help avoid routine lymphadenectomies without compromising oncological safety. The procedure is simple and can be practiced at all centres and can also aid pathologists by pinpointing the likely metastatic nodes after a selective or complete lymphadenectomy.     

荧光染色技术在艾滋病患者活检标本病原学诊断中的应用

目的探讨快速真菌与抗酸分枝杆菌荧光技术在艾滋病患者活检标本病原体诊断中的应用价值及意义。方法选取艾滋病患者500例活检标本,采用快速真菌荧光染色及六胺银、PAS染色对300例可疑真菌感染的活检标本进行检测,采用抗酸分枝杆菌荧光染色及萋尼抗酸染色对200例可疑结核的活检标本进行检测,并对检测结果进行对比。结果（1）快速真菌荧光镜检法检出阳性205例（阳性率68.3%）,六胺银染色法检出阳性209例（阳性率69.7%）,PAS染色法检出阳性97例（阳性率39.5%）,快速真菌荧光染色与六胺银染色检出阳性率差异无统计学意义（P>0.05),但高于PAS染色法;（2）抗酸分枝杆菌荧光染色检出阳性179例（阳性率89.5%）,萋尼抗酸染色法检出阳性117例（阳性率58.5%）,抗酸分枝杆菌荧光染色阳性率高于萋尼抗酸染色法,两者差异有统计学意义（P<0.01）;（3）荧光染色比常规特殊染色操作更快速、简便、显示更清晰。结论艾滋病患者并发机会性感染病原体种类多样,普通的特殊染色阳性率不高并且费时费力,而快速真菌及抗酸荧光染色特别是抗酸荧光染色的阳性率明显增高,为活检组织诊断真菌及结核分枝杆菌感染提供一种快速、准确、经济、对比更清晰的染色方法,能够为真菌感染及结核病患者诊断提供可靠的指导依据,值得在病原体诊断工作中广泛使用。