癌细胞分析系统的研制

癌细胞分析系统的研制＊高敦岳陆宗骐张建正张雷（华东理工大学自动化所上海２００２３７）０引言癌症是严重危害人类健康的恶性病，及时准确地诊断癌症至关重要。至今，国内癌症的诊断仍采用一百多年以来的常规的细胞病理形态定性分析，具有很大主观性，容易误诊。本文研...

天花粉蛋白诱导肝癌Ⅱ_(ZZ)瘤株凋亡的实验研究

目的:探讨天花粉蛋白(TCS)诱导肝癌ⅡZZ瘤株凋亡的作用机制。方法:采用不同浓度的TCS处理细胞后,用流式细胞仪检测肝癌细胞ⅡZZ瘤的凋亡率及细胞周期分布。结果:50μmol/L、100μmol/L的TCS能诱导肝癌细胞ⅡZZ瘤凋亡,凋亡率与剂量呈正相关;ⅡZZ随药物浓度及作用时间变化,细胞周期分布呈现G1期细胞比例逐渐增高,并出现典型的凋亡峰。结论:TCS具有一定的抗肝癌作用,其机制与促进肝癌细胞凋亡及抑制癌细胞增殖有关。

The Clinical Pathologic Analysis of Radiotherapy for Cutaneous B-cell Lymphoma

OBJECTIVE To report results of radiation therapy treatment of 30 B-cell lymphoma patients with an initial cutaneous presentation according to the new classification by the WHO/EORTC. METHODS Thirty patients with cutaneous B-cell lymphoma （CBCL） were treated by cutaneous irradiation based on the number and location of the lesions and the stage of their tumor. Treatment was conducted using a Satume Clinac. RESULTS A complete response （CR） from the treatment for our series was 86%. The length of complete remission ranged from 4 to 301 months. Three patients （11%） developed a partial response （PR）. One patient was progressive. Disease-free survival（DFS） at 10 years was 87%. Three patiens died [One PCMZL two PCLBCL leg type （29%）]. Radiotherapy was generally well tolerated. CONCLUSION According to the WHO/EORTC classification, the survivor results were good for PCMZL and PCFCL. The PCLBCL leg type had a poor prognosis. Localized field irradiation is an effective treatment for some localized forms of primary cutaneous B-cell lymphoma, and this mode of therapy can produce prolonged remissions.The patients with wide-spread skin involvement are usually candidates for extended field irradiation and/or chemotherapy. For advanced stages of cutaneous B-cell lymphoma, where chemotherapy is the treatment of choice, a degree of palliation can be achieved using local field irradation.

Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

AIM： To explore the effect of silencing of signal transducer and activator of transcription 3 （STAT3） expression by RNA interference （RNAi） on growth of human hepatocellular carcinoma （HCC） in tumorbearing nude mice in vivo.METHODS： To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP （pSHI-siRNA- STAT3） and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSHI-siRNA- STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction （RT- PCR）. Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay was used to detect apoptosis of tumor cells.RESULTS： The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups （P 〈 0.01）. The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased （P 〈 0.01）. Most of the tumor tissue ceils in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION： Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem （ES） cells and induced pluripo- tent stem （iPS） cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDXl-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insuIin-producing ceils. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.

Shenqi Fuzheng injection combined with GP chemotherapy in the treatment of advanced non-small cell lung cancer： a meta-analysis

Objective： To evaluate the clinical efficacy of Shenqi Fuzheng injection combined with gemcitabine plus cisplatin（GP） in the treatment of advanced non-small cell lung cancer （NSCLC）. Methods： we performed a systematicsearch in the electronic databases such as Cochrane Library, Pubmed, Embase, Chinese Journal Full-text Database,Chinese Biomedical Literature Database, Chinese Science and Technology Periodical Full-text Database andWanfang Database up to 30 January 2017. Randomized controlled trials （RCT） of Shenqi Fuzheng Injectioncombined with GP chemotherapy in the treatment of advanced NSCLC were searched, and all the RCTs wereconducted on methodological quality assessment. Data extraction and data analysis were according to standards ofCochrane systematic review. Results： Eight trials were included including a total of 701 patients. Meta-analysisresults： Shenqi Fuzheng injection combined with GP chemotherapy could significantly improve the functionalstatus of patients with NSCLC （OR = 3.44, 95% CI [2.26, 5.25], P 〈 0.0001） and clinical treatment efficacy （OR =（OR = 0.31, 95%CI [0.20, 0.47], P 〈 0.0001. The rate of leukopenia （OR = .31, 95%CI [0.20,0.47], P 〈 0.0001）,thrombocytopenia （OR = 0.58, 95%CI [0.37, 0.91], P = 0.020）, hemoglobin decline （（OR = 0.31, 95%CI [0.16,0.59], P = 0.0004） and incidence of gastrointestinal reactions （OR = 0.58,P 〈 0.05） could be reduced. Conclusion：Shenqi Fuzheng injection combined with GP chemotherapy in the treatment of advanced NSCLC obtainedsignificantly clinical efficacy. The quality of the literature incorporated is low, the conclusion requires high-qualityresearch to further prove.

Microarray-based analysis for hepatocellular carcinoma: From gene expression profiling to new challenges

Accumulation of mutations and alterations in the expression of various genes result in carcinogenesis,and the development of microarray technology has enabled us to identify the comprehensive gene expression alterations in oncogenesis.Many studies have applied this technology for hepatocellular carcinoma(HCC),and identified a number of candidate genes useful as biomarkers in cancer staging,prediction of recurrence and prognosis,and treatment selection.Some of these target molecules have been used to develop new serum diagnostic markers and therapeutic targets against HCC to benefit patients.Previously,we compared gene expression profiling data with classification based on clinicopathological features,such as hepatitis viral infection or liver cancer progression.The next era of gene expression analysis will require systematic integration of expression profiles with other types of biological information,such as genomic locus,gene function,and sequence information.We have reported integration between expression profiles and locus information,which is effective in detecting structural genomic abnormalities,such as chromosomal gains and losses,in which we showed that gene expression profiles are subject to chromosomal bias.Furthermore,array-based comparative genomic hybridization analysis and allelic dosage analysis using genotyping arrays for HCC were also reviewed,with comparison of conventional methods.

Clinical observation of gemcitabine and concomitant three-dimensional conformal radiotherapy in the treatment of locally advanced non-small cell lung cancer

Objective： To evaluate the clinical effect of gemcitabine and concurrent three-dimensional conformal radiation therapy （3D-CRT） for locally advanced non-small cell lung cancer （NSCLC）. Methods： From April 2002 to June 2005, 38 patients with inoperable stage Ⅲ NSCLC were treated with gemcitabine and 3D-CRT simultaneously. Chemotherapy consisted of intravenously gemcitabine 350 mg/m^2 on days 1, 8, 15, 22, 29, 36.3D-CRT was delivered up to a total dose of 60-64 Gy with a 2.0 Gy dose fraction per day, 5 days per week. Results： The overall response rates of primary tumor and mediastinum metastatic node were 86.8% （33/38） and 90.6% （29/32） respectively, and 91.7% （22/24） and 78.6% （11/14） for squamous cell carcinoma and adenocarcinoma respectively. The acute side effects of patients were mostly myelosuppression, nausea, vomiting, radiation-induced esophagitis and pneumonitis （RTOG 1/11）, however, all of them were cured. Conclusion： Concurrent application of gemcitabine and 3D-CRT can improve the overall response rate for locally advanced NSCLC without aggravating the side effects.

Single-cell analyses of circulating tumor cells

Circulating tumor cells(CTCs) are a population of tumor cells mediating metastasis, which results in most of the cancer related deaths. The number of CTCs in the peripheral blood of patients is rare, and many platforms have been launched for detection and enrichment of CTCs. Enumeration of CTCs has already been used as a prognosis marker predicting the survival rate of cancer patients. Yet CTCs should be more potential. Studies on CTCs at single cell level may help revealing the underlying mechanism of tumorigenesis and metastasis. Though far from developed, this area of study holds much promise in providing new clinical application and deep understanding towards metastasis and cancer development.

Screening serum hepatocellular carcinoma-associated proteins by SELDI-based protein spectrum analysis

AIM： To find out potential serum hepatocellular carcinoma （HCC）-associated proteins with low molecular weight and low abundance 13y SELDI-based serum protein spectra analysis, that will have much application in the diagnosis or differentiated diagnosis of HCC, as well as giving a better understanding of the mechanism of hepato-card nogenesis.METHODS： Total serum samples were collected with informed consent from 81 HCC patients with HBV（＋）/ cirrhosis（＋）, 36 cirrhosis patients and 43 chronic hepatitis B patients. Serum protein fingerprint profiles were first generated by selected WCX2 protein chip capture integrating with SELDI-TOF-MS, then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard..Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra, some protein peaks with significant difference between H.CC and cirrhosis or chronic hepatitis B were found.RESULTS： One hundred and twenty-eight serum protein peaks betweeri.2000 and 30000Da were identified under the condition of signal-to-noise 〉 5 and minimum threshold for cluster 〉 20%. Eighty-seven of these proteins were showed significant differences in intensity between HCC and cirrhosis （P 〈 0.05）. Of the above differential proteins, 45 proteins had changes greater than two-fold, including 15 upregulated proteins and 30 downregulated proteins i.n HCC serum. Between HCC and chronic hepatitis B, 9 of 52 differential proteins （P 〈 0.05） had intensities of more than two-fold, including 2 upregulated proteins and 7 downregulated proteins in HCC serum. Between cirrhosis and chronic hepatitis B, 28 of 79 significant differential proteins （P 〈 0.05） changes greater than two-fold in intensity, including 17 upregulated proteins and 11 downregulated proteins in cirrhosis serum. For the analysis of these leading differential proteins in subtraction difference mode among three diseases, the five common downregulated proteins in HCC serum （M/Z 2870, 3941, 2688, 3165, 5483） and two common upregulated proteins （M/Z 3588, 2017） in HCC and cirrhosis serum were screened.CONCLUSION： Because the interference of unspecific secreted proteins from hepatitis B and cirrhosis could be eliminated partly in HCC serum under subtraction difference analysis, these seven common differential proteins have the obvious advantage of specificity for evaluating the pathological state of HCC and might become novel candidate biomarkers in the diagnosis of HCC.

Mechanism of induction apoptosis of Onychin in ovarian cells in vitro

Objective： The aim of the study was to investigate the possible mechanism of induction apoptosis of Onychin （ONY） in ovarian cancer HO-8910 cells in vitro. Methods： Human ovarian cancer HO-8910 cells were cultured in vitro. Inhibi- tory effect of ONY on the viability of HO-8910 cells was evaluated by the MTT assay. Apoptosis of HO-8910 cells treated with different concentrations of ONY for 48 h was detected by FCM. Expression of proteins related to apoptosis was analyzed by Western blot. Results： ONY significantly inhibited the viability of human ovarian cancer HO-8910 cells in a dose-dependent and time-dependent manner, and the ICso was 10.48 pg/mL for 48 h. The cells treated with ONY showed typical morphological change of apoptosis and increased cells of sub-G1 population by FCM in a dose-dependent. Western blot showed that ex- pression of Bax, cytochrome C, caspase-9 and caspase-3 proteins were upregulated and protein level of Bcl-2 was depressed after treatment with ONY in a concentration dependent. Conclusion： Apoptosis of ovarian cancer HO-8910 cells was induced by ONY through mitochondrial apoptosis pathway in vitro.

Quantitative analysis using ELISA of vascular endothelial growth factor and basic fibroblast growth factor in human colorectal cancer,liver metastasis of colorectal cancer and hepatocellular carcinoma

Angiogenesis consists of the sprouting of capillaries from pre-existing vessels. It is well-known that tumor growth is angiogenesis-dependent. Vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） stimulated vascular endothelial cell proliferation and are involved in the neoplastic angiogenesis of several types of tumors including those of the intestinal tract. Authors usually investigated VEGF and using immunohistochemistry bFGF protein expressions or Western blotting and VEGF and bFGF transcripts using reverse transcriptase Dolymerase chain reaction （RT-PCR）.

Clear cell adenocarcinoma of the colon is a unique morphological variant of intestinal carcinoma:Case report with molecular analysis

Here we report a new case of clear cell adenocarcinoma (CCA) of the colon in a 54-year-old Caucasian man. Despite of the previous reported cases, the lesion was located in the right colon and was not associated with the conventional adenoma. We performed immunohistochemical and molecular analyses in order to explore whether the CCA had the molecular features generally associated with conventional colorectal carcinoma. The immunohistochemical and molecular analyses showed that the different morphology of CCA does not reflect a distinct biological entity but only an unusual morphological variant of intestinal carcinoma.

Immune profiling and cancer post transplantation

Half of all long-term(> 10 year) australian kidney transplant recipients(KTR) will develop squamous cell carcinoma(SCC) or solid organ cancer(SOC), making cancer the leading cause of death with a functioning graft. At least 30% of KTR with a history of SCC or SOC will develop a subsequent SCC orSOC lesion. Pharmacological immunosuppression is a major contributor of the increased risk of cancer for KTR, with the cancer lesions themselves further adding to systemic immunosuppression and could explain, in part, these phenomena. Immune profiling includes; measuring immunosuppressive drug levels and pharmacokinetics, enumerating leucocytes and leucocyte subsets as well as testing leucocyte function in either an antigen specific or non-specific manner. Outputs can vary from assay to assay according to methods used. In this review we define the rationale behind post-transplant immune monitoring assays and focus on assays that associate and/or have the ability to predict cancer and rejection in the KTR. We find that immune monitoring can identify those KTR of developing multiple SCC lesions and provide evidence they may benefit from pharmacological immunosuppressive drug dose reductions. In these KTR risk of rejection needs to be assessed to determine if reduction of immunosuppression will not harm the graft.

Hepatocellular carcinoma and multidrug resistance: Past, present and new challenges for therapy improvement

Hepatocellular carcinoma(HCC) is the most frequent form of liver cancer and the third most common cause of cancer-related death in the world. The main risk factor worldwide for this type of malignancy is chronic hepatitis caused by hepatitis B virus and hepatitis C virus infections. Advances in early detection and treatmenthave improved life expectancy of patients with HCC. However, this disorder remains as a disease with poor prognosis. In fact, epidemiological studies have revealed that there is an 8-mo median survival rate in patients, approximately 20% of whom survive one year while only 5% remain alive after three years. Additionally, HCC is particularly difficult to treat because of its high recurrence rate, and its resistance to conventional chemotherapy is due, among other mechanisms, to several members of the ATP-Binding Cassette protein family involved in drug transport being overexpressed. Fortunately, there is evidence that these patients may benefit from alternative molecular-targeted therapies. This manuscript intends to provide further insight into the etiology and molecular mechanisms related to HCC development and the latest therapeutic approaches to treat this malignancy. The development of effective delivery systems of antitumor drugs able to target the liver parenchyma is also assessed. Finally, the prospects in the development of more efficient drug therapies to overcome multidrug resistance are also examined.

Is there a role for systemic targeted therapy after surgical treatment for metastases of renal cell carcinoma?

Metastatic renal cell carcinoma(m RCC) is a challenging disease. Despite the new targeted therapies, complete remissions occur only in 1%-3% of the cases, and the most effective first-line treatment drugs have reached a ceiling in overall survival(ranging from 9 to 49 mo). Metastasectomy remains to be the only curative option in most patients with m RCC. Prognostic nomograms have been recently published, so we have tools to classify patients in risk groups, allowing us to detect the cases with the higher risk of recurrence after metastasectomy. Although sparse, there is some evidence of effectiveness of neoadjuvant targeted therapy before metastasectomy; but with an increase in surgical complications due to the effects of these new drugs in tissue healing. We have aimed to answer the question: Is there a role for systemic targeted therapy after surgical treatment for metastases of renal cell carcinoma? We have made a search in Pubmed database. As far as we know, evidence is low and it's based in case reports and small series of patients treated with adjuvant drugs after neoadjuvant therapy plus metastasectomy in cases of partial response to initial systemic treatment. Despite the limitations and high risk of bias, promising results and cases with longterm survival with this approach have been described. Two ongoing clinical trials may answer the question that concerns us.

Changes of NF-κB, Bax and Caspase 3 in Apoptosis Induced by Ligustrazine Combined with Cis-dichlorodiamine Platinum in Human Gastric Carcinoma SGC-7901 Cell Lines

[Objective] This study aimed to investigate the mechanism of apoptosis induced by ligustrazine(TMP) and cis-dichlorodiamine platinum(DDP) in SGC-7901 cell lines in vitro. [Methods] SGC-7901 cell lines were treated with ligustrazine and DDP alone or combined for 48 h for Western blot analysis, respectively. Western blot analysis was used to determine the expression of proteins involved in apoptosis including NF-κB p65, bax and caspase-3. [Results] The viability of SGC-7901 cells was inhibited after treated with ligustrazine and/or combined with DDP. The expression of NF-κB P65 protein decreased after treated with drugs, in which the protein decreased significantly in 1.2 mg/ml of TMP combined with 2 μg/ml of DDP group.Meanwhile, we investigated the protein expression of bax and caspase-3. The results showed that the expression of the two proteins increased following with the increasing concentration of TMP. [Conclusion] All the results indicated that ligustrazine combined with DDP could induce the apoptosis of SGC-7901 cell lines, and NF-κB maybe the possible way to induce the cell apoptosis.

Risk factors for residual tumor after resection of hepatocellular carcinoma

AIM:To identify the clinicopathological risk factors correlated with residual tumor in hepatocellular carcinoma (HCC) patients after resection. METHODS:From January 2001 to April 2007,766 HCC patients who had undergone resection were included in this research. Lipiodol angiography was performed within 2 mo after surgery and followed by post-Lipiodol computed tomography (CT) 4 wk later for all 766 patients to monitor tumor in the remnant liver. Tumor detected within the first 3-mo postoperative period was defined as residual tumor. Patients were divided into 2 groups:disease or disease-free within the first 3 mo after surgery. Risk factors for residual tumor were investigated among various clinicopathological variables. RESULTS:A total of 63 (8.22%) patients were found to have residual tumor after surgery. Three independent factors associated with residual tumor were identified by multivariate analysis:preoperative serum α-fetoprotein (AFP) level [odds ratio (OR) = 1.68 (95% confidence interval (CI):1.20-2.36)],tumor size [OR = 1.73 (95% CI:1.29-2.31)] and microvascular invasion [OR = 1.91 (95% CI:1.12-3.24)]. CONCLUSION:Residual tumor is related to AFP level,tumor size and microvascular invasion. Patients at high risk should undergo closer follow-up and could be candidates for multimodality therapy.

MR-arterioportography: A new technical approach for detection of liver lesions

AIM: To evaluate the benefit and effectiveness of MRarterioportography (MR-AP) to achieve the highest sensitivity for detection and evaluation of hepatocellular carcinoma (HCC). METHODS: Twenty liver cirrhosis patients with suspected HCC were included before transarterial chemoembolization. In all patients double-enhanced Magnetic resonance imaging (MRI) was performed. A bolus of 10 mL Magnevist was injected through a selectively placed catheter in the superior mesenteric artery and MRI of the liver was performed in arterioportographic phase. Two independent readers evaluated number, size and localization of detected lesions. Diagnostic quality was determined using a 4-point scale. Differences were analyzed for significance using a t -test. Interobserver variability was calculated. RESULTS: In all 20 patients (100%), MR-AP was feasible. Diagnostic quality was, in all cases, between 1 and2 for both modalities and readers. MR-AP detected significantly more lesions than double-enhanced MRI (102.5 vs 61, respectively, P < 0.0024). The inter-observer variability was 0.881 for MRI and 0.903 for MR-AP. CONCLUSION: Our study confirmed that the MR-AP as an additional modality for detection of HCC is beneficial, as significantly more lesions were detected compared to MRI with liver-specific contrast.