Detection of peripheral blood circulating tumor cells in oral squamous cell carcinoma and its clinical significance.

OBJECTIVES: This study aims to investigate the diagnostic value of peripheral blood circulating tumor cells (CTCs) in oral squamous cell carcinoma (OSCC) and its correlation with the clinicopathological features of OSCC. METHODS: Ninety-three patients diagnosed as OSCC in the First Affiliated Hospital of Zhengzhou University from May 2019 to May 2020 were selected as the experimental group, and 20 healthy volunteers were employed as the control group. The CTCs value of peripheral blood of the patients were measured by CTCs detection technology, and its clinical significance was analyzed. RESULTS: The CTCs values in the experimental group were higher than those in the control group, and the difference was statistically significant (P<0.000 1). The CTCs value in the peripheral blood of patients in the experimental group were not correlated with gender, site of onset, and presence or absence of peripheral tissue infiltration (P>0.05), but was correlated with age (P=0.022), tumor T stage (P=0.02), tumor N stage (P=0.007 5), tumor M stage (P=0.013), clinical stage (P=0.029), early or late stage (P=0.022), tumor differentiation degree (P<0.001), and node metastasis (P=0.006 4). The AUC value of CTCs in OSCC diagnosis was 0.925, and the energy efficiency was statistically significant [P=0.000, 95%CI (0.876, 0.974)]. When the CTC value was 8.450 FU/3 mL, the maximum value of the Yoden index was 0.853, and the sensitivity and specificity of OSCC diagnosis were 90.3% and 95.0%, respectively. The AUC value of CTCs in the diagnosis of OSCC metastasis was 0.691, and the energy efficiency was statistically significant [P=0.000, 95%CI (0.580, 0.803)]. When the blood CTC value was 12.250 FU/3 mL, the maximum value of Yoden index was 0.367, the sensitivity was 63.6%, and the specificity was 73.3%. Multivariate regression analysis showed that buccal tumor was negatively correlated with CTCs in patients with OSCC (P=0.001 08), N2 stage (P=0.000 74) and M stage (P=0.026 38). High differentiation (P<0.000 1) and moderate differentiation (P=0.001 5) were negatively correlated with CTCs values in patients with OSCC. CONCLUSIONS: Peripheral blood CTCs has important clinical value for early screening, auxiliary diagnosis, evaluation of metastasis, and determination of malignant degree, progression, and pathological grade of OSCC and a relatively reliable tumor detection indicator.

Multifocal glioblastoma-two case reports and literature review.

BACKGROUND: Multifocal glioblastoma is a rare type of glioblastoma with worse prognosis. In this article, we aimed to report two cases of classical multifocal glioblastoma. CASE PRESENTATION: In case 1, a 47-year-old male presented with dizziness, and once had a sudden loss of consciousness accompanied by convulsion of limbs. Contrast-enhanced MRI showed multiple lesions with heterogeneously ring-enhanced characters in the left hemisphere, diagnosed as multifocal glioblastoma. He underwent a craniotomy of all lesions, concurrent radiotherapy and chemotherapy as well as additional chemotherapy of temozolomide. After 2 cycles, repeat MRI showed that the new lesions already occurred and progressed. Eventually, he abandoned the chemotherapy after the 2 cycles and died 1 year later. In case 2, a 71-year-old male presented with a history of headache, left limb weakness, and numbness. Discontinuous convulsion of limbs once occurred. Contrast-enhanced MRI showed multiple lesions located in the right hemisphere, diagnosed as multifocal glioblastoma. He underwent a right frontoparietal craniotomy of the main lesion. Hemorrhage of the residual tumor and pulmonary artery embolism occurred synchronously. Eventually, his family decided not to pursue any further treatment and opted for hospice care and he passed away within 11 days of surgery. CONCLUSIONS: We reported two cases of typical multifocal glioblastoma. Valid diagnosis is crucial; then, resection of multiple lesions and canonical radio-chemotherapy probably bring survival benefits.

Safety and efficacy of INTRABEAM intraoperative radiotherapy for invasive thymoma.

Intraoperative radiotherapy (IORT) has been used to treat different residual solid tumors after tumor removal and has shown many advantages over other treatment methods. However, the use of IORT for invasive thymoma has not been reported. Therefore, in this study, we tried to determine the safety and efficacy of INTRABEAM IORT for the treatment of invasive thymoma.Among the patients admitted to our hospital from September to December 2016 who were diagnosed with invasive thymoma, 14 were selected as study subjects. With medical histories taken beforehand, 8 of these patients were diagnosed with Masaoka stage IIA and 6 with Masaoka stage IIB; furthermore, 5 of the patients were diagnosed with myasthenia gravis (MG). INTRABEAM radiation (8-10 Gy, low energy) was delivered to the postoperative tumor bed of each patient during surgery. The intra- and postoperative complications were observed and evaluated, and the improvement in symptoms was assessed. An additional 23 patients with stage II thymoma undergoing radical surgery from April to August 2016 were chosen as the control group.One month after the operation, only 1 patient in the IORT group had cough, increased levels of leucocytes and neutrophils, and pulmonary inflammation on chest computed tomography. Reactive inflammation and pleural effusion in the 2 groups were similar (P > .05). There was no significant difference between the 2 groups in the improvement of myasthenia gravis (P > .05). Postoperative chest computed tomography and routine blood examination at 3 and 12 months showed that all the patients recovered, with normal hemogram levels and no pulmonary fibrosis around the radiation field. In addition, ultrasonic cardiography and electrocardiography demonstrated no significant difference before or after surgery within the IORT group. At the end of the follow-up, all the patients were alive, no relapse or remote metastasis was observed in the IORT group, and 2 inpatients in the control group had experienced relapse at 24 and 26 months. There was a significant difference in disease-free survival between the 2 groups (P = .00).It is safe to administer low-energy INTRABEAM IORT at a dose of approximately 10 Gy in patients with stage II invasive thymoma. INTRABEAM IORT does not significantly increase operation- or radiation-related complications and has no significant effect on vital organs such as the lungs and heart. Its long-term efficacy is worth expecting.

Long non-coding RNA CCAT1 is a prognostic biomarker for the progression of oral squamous cell carcinoma via miR-181a-mediated Wnt/ß-catenin signaling pathway.

Oral squamous cell carcinoma (OSCC) ranks as the sixth most common carcinoma worldwide, and the third most common carcinoma in developing countries as well. Recently, the aberrant expression of lncRNA CCAT1 has been revealed to play an important role in the development of several cancers. However, its role in OSCC remains unknown. The expression levels of CCAT1 and miR-181a were determined in 15 paired primary OSCC tissues and their adjacent noncancerous tissues and cell lines with qPCR. shRNA against CCAT1 was employed to investigate the impact of CCAT1 on proliferation and metastasis. Then dual luciferase reporter and RIP assays were utilized to study the interaction between CCAT1 and miR-181a. Cells transfected with sh-CCAT1 or treated with miR-181a inhibitor were subjected to western blot to investigate the role of Wnt/ß-catenin signaling in CCAT1-mediated proliferation and metastasis. Finally, the role of CCAT1 in OSCC was confirmed with tumor xenografts mice model. CCAT1 was upregulated in OSCC tissues and cell lines. Knockdown of CCAT1 inhibited the proliferation, migration and invasion of OSCC cells, while the cell apoptosis was enhanced. Luciferase and RIP assays revealed that miR-181a was a direct target of CCAT1. Inhibition of miR-181a partially reversed the efficacy of sh-CCAT1. Moreover, sh-CCAT1 inhibited OSCC tissues growth through inhibiting Wnt signaling in a miR-181a-dependent manner in vivo. lncRNA CCAT1 activated Wnt/ß-catenin signaling via inhibiting miR-181a, resulting in the cell proliferation, migration and invasion of OSCC, suggesting that CCAT1 might serve as a potential target of OSCC treatment. Abbreviation: LncRNA: long non-coding RNA; OSCC: oral squamous cell carcinoma; 3' UTR: 3' untranslated region; ANOVA: one-way analysis of variance; CDK: cyclin-dependent kinase; ceRNA: competing endogenous RNA; FBS: fetal bovine serum; HGF: human gingival fibroblasts; MAPK: mitogen-activated protein kinase; miRNA: micro RNA; ncRNA: noncoding RNAs; PBS: phosphate-buffered saline; PI3K: phosphatidylinositol 3-kinase.

Construction of Anti-hPD-L1 HCAb Nb6 and in Situ 124I Labeling for Noninvasive Detection of PD-L1 Expression in Human Bone Sarcoma.

Immunotherapy is considered the fourth major treatment mode for cancer following surgery, chemotherapy, and radiotherapy. In recent years, tumor immunotherapy has achieved breakthrough progress; therefore, it is important to screen patients to identify those who will respond to tumor immunotherapy. Here, we report the construction of a novel heavy chain-only antibody (HCAb) and its corresponding 124I-labeled probe. Using phage display technology, we generated a novel anti-hPD-L1-specific HCAb named Nb6 (selected from 95 monoclones) with high affinity for hPD-L1. The positron-emitting 124I-labeled hPD-L1-targeted HCAb probe was prepared for further evaluation, and nonradioactive natural iodine (natI)-labeled anti-hPD-L1 Nb6 was synthesized as a reference compound. 125I-anti-hPD-L1 Nb6 uptake in OS-732 cells in vitro can be blocked by the precursor. The binding affinity of 125I-anti-hPD-L1 Nb6 to OS-732 cell lines was 2.19 nM. For in vivo studies, an osteosarcoma OS-732 tumor-bearing mouse model was successfully constructed. Polymerase chain reaction (PCR) and Western blot analyses were performed to confirm the presence of the hPD-L1 gene and antigen in the tumor tissue of the OS-732 mouse model. Biodistribution showed that uptake of 124I-anti-hPD-L1 Nb6 probes at 24 h was 4.43 ± 0.33% ID/g in OS-732 tumor tissues. Tumor lesions can be clearly delineated on micro-PET (positron emission tomography)/CT (computed tomography) imaging 24 h after injection of 124I-anti-hPD-L1 Nb6, while the blocking group shows substantially decreased uptake on imaging. Pathological staining validated hPD-L1 expression on the surface of the tumor cell membrane; thus, 124I-anti-hPD-L1 Nb6 can be used for in vivo noninvasive PET imaging. When administered in tandem, Nb6 and 124I-anti-hPD-L1 Nb6 may provide a novel strategy to clinically screen patients for hPD-L1 to identify those who would benefit from immunotherapy of malignant tumors such as osteosarcoma.

MCLPMDA: A novel method for miRNA-disease association prediction based on matrix completion and label propagation.

MiRNAs are a class of small non-coding RNAs that are involved in the development and progression of various complex diseases. Great efforts have been made to discover potential associations between miRNAs and diseases recently. As experimental methods are in general expensive and time-consuming, a large number of computational models have been developed to effectively predict reliable disease-related miRNAs. However, the inherent noise and incompleteness in the existing biological datasets have inevitably limited the prediction accuracy of current computational models. To solve this issue, in this paper, we propose a novel method for miRNA-disease association prediction based on matrix completion and label propagation. Specifically, our method first reconstructs a new miRNA/disease similarity matrix by matrix completion algorithm based on known experimentally verified miRNA-disease associations and then utilizes the label propagation algorithm to reliably predict disease-related miRNAs. As a result, MCLPMDA achieved comparable performance under different evaluation metrics and was capable of discovering greater number of true miRNA-disease associations. Moreover, case study conducted on Breast Neoplasms further confirmed the prediction reliability of the proposed method. Taken together, the experimental results clearly demonstrated that MCLPMDA can serve as an effective and reliable tool for miRNA-disease association prediction.

[miR-199b-5p inhibits proliferation and invasion of head and neck cancer cells].

PURPOSE: This study identified the differentially expressed miR-199b-5p in the progressing of head and neck squamous cell carcinoma(HNSCC) and investigated its biological characters. METHODS: The expression of miR-199b-5p in 128 HNSCC tissues samples was evaluated. The association between clinicopathological parameters and the expression levels of the candidated miRNAs was analyzed by using Kaplan-Meier survival analysis. Cell growth, invasion and migration potential, and clone formation were observed to detect the functions of the miRNAs in HNSCC cells. Statistical analysis was performed using SPSS 19.0 software package. RESULTS: In 79 HNSCC tissues with cervical lymph node metastasis, the expression level of miR-199b-5p was 1.68±0.21; while the expression level was 2.64±0.24 in 28 tissues without lymph node metastasis (P=0.001). In patients with HNSCC, lower level of miR-199b-5p expression significantly correlated with worse overall survival rate (P=0.01). Overexpression of miR-199b-5p inhibited HNSCC cell proliferation and invasion. CONCLUSIONS: miR-199b-5p plays a key role in cell invasion and metastasis and its expression correlated with overall survival in patients with HNSCC.

GLNMDA: a novel method for miRNA-disease association prediction based on global linear neighborhoods.

Recently, increasing studies have shown that miRNAs are involved in the development and progression of various complex diseases. Consequently, predicting potential miRNA-disease associations makes an important contribution to understanding the pathogenesis of diseases, developing new drugs as well as designing individualized diagnostic and therapeutic approaches for different human diseases. Nonetheless, the inherent noise and incompleteness in the existing biological datasets have limited the prediction accuracy of current computational models. To solve this issue, in this paper, we propose a novel method for miRNA-disease association prediction based on global linear neighborhoods (GLNMDA). Specifically, our method obtains a new miRNA/disease similarity matrix by linearly reconstructing each miRNA/disease according to the known experimentally verified miRNA-disease associations. We then adopt label propagation to infer the potential associations between miRNAs and diseases. As a result, GLNMDA achieved reliable performance in the frameworks of both local and global LOOCV (AUCs of 0.867 and 0.929, respectively) and 5-fold cross validation (average AUC of 0.926). Case studies on five common human diseases further confirmed the utility of our method in discovering latent miRNA-disease pairs. Taken together, GLNMDA could serve as a reliable computational tool for miRNA-disease association prediction.

[Role and mechanism of Galectin-3 gene in proliferation, invasion, and apoptosis of oral squamous cell carcinoma].

OBJECTIVE: The effect and mechanism of Galectin-3 gene expression on proliferation, invasion, and apoptosis of oral squamous cell carcinoma (OSCC) were investigated. METHODS: Reverse transcription-polymerase chain reaction (RT-
PCR) and Western blotting were used to detect the mRNA and protein of Galectin-3 gene in OSCC. OSCC Tca8113 was divided into control, negative control, and Galectin-3 transfection groups. Western blotting was used to detect the expression of Galectin-3, matrix metalloproteinase (MMP)-2, MMP-9, Cleaved Caspase-3, ß-catenin, and Cyclin D1 protein after transfection for 48 h in each group. Cell proliferation was detected by CCK8. Cell invasion ability was detected by using a Transwell chamber. Cell apoptosis was detected by flow cytometry. RESULTS: The mRNA and protein expression levels of Galectin-3 gene in OSCC were significantly higher than those in adjacent tissues (P<0.01). Galectin-3 protein expression in Tca8113 cells significantly decreased after RNA interference. Cell survival rate and invasion as well as MMP-2, MMP-9, ß-catenin, and Cyclin D1 protein expression were significantly lower than the blank group. Apoptosis rate and Cleaved Caspase-3 protein expression were significantly higher than the control group (P<0.01). CONCLUSIONS: Inhibition of Galectin-3 gene expression in OSCC can significantly reduce the proliferation and invasion of cancer cells and induce apoptosis. The mechanism is related to downregulation of the Wnt/ß-catenin signaling pathway.

MicroRNA-16 functions as a tumor-suppressor gene in oral squamous cell carcinoma by targeting AKT3 and BCL2L2.

Aberrant expressions of microRNAs have been reported to be strongly associated with the progression and prognosis of various tumors, including oral squamous cell carcinoma (OSCC). Recent studies on miRNA expression profiling have suggested that microRNA-16 (miR-16) may be dysregulated in OSCC. However, the tumorigenic roles and mechanisms of miR-16 in OSCC are still largely unknown. In this study, we demonstrated that miR-16 was specifically downregulated in both OSCC patients and cancer cell lines. In addition, functional roles of miR-16 in vitro suggested that the miR-16 mimic inhibited cell proliferation and induced apoptosis, whereas miR-16 inhibitor displayed the opposite effects. Luciferase reporter assay and correlation analysis showed that AKT3 and BCL2L2 were directly targeted by miR-16 and were inversely expressed with miR-16 in OSCC. Moreover, restoration of AKT3 and BCL2L2 expression could partially reverse the cell proliferation inhibition and apoptosis induction caused by miR-16. In xenograft nude mice, miR-16 mimics decreased the expression of AKT3 and BCL2L2 and reduced the tumors volumes and weights, whereas the miR-16 inhibitor exhibited adverse effects in the derived xenografts. In conclusion, the findings suggested that miR-16 functions as a tumor suppressor miRNA to inhibit cell proliferation and induce apoptosis in OSCC through decreasing the oncogenes AKT3 and BCL2L2 and that miR-16 could be a potential therapeutic target for OSCC.

Inhibition of EGR1 inhibits glioma proliferation by targeting CCND1 promoter.

BACKGROUND: Gliomas are the most common primary tumors in central nervous system. The prognosis of the patients with glioma is poor regardless of the development of therapeutic strategies. Its aggressive behavior mainly depends on the potent ability of proliferation. The transcription factor EGR1 (early growth response 1) is a member of a zinc finger transcription factor family which plays an essential role in cell growth and proliferation. METHODS: EGR1 expression levels in 39 glioma tissues and 10 normal brain tissues were tested by RT-qPCR and Western-blotting. The effects of EGR1 on U251 cells, U251 stem-like cells (GSCs), and U87 cells proliferation were assessed using in vitro and in vivo cell proliferation assays. The specific binding between EGR1 and CCND1 promoter was confirmed by CHIP assay. EGF was used to improve EGR1 expression in this assay. RESULTS: EGR1 expression levels in human gliomas are decreased compared with normal brain tissues, however, the patients with low EGR1 expression level showed significantly enhanced patient survival in all glioma patients. EGR1 silencing inhibited proliferation and induced G1 phase arrest in glioma cells. EGR1 contributed to proliferation by directly raising CCND1. Meanwhile, EGR1 overexpression induced by EGF was able to promote the proliferation of glioma cells. CONCLUSIONS: Our results show that stable knockdown EGR1 would inhibit glioma proliferation. The results suggest EGR1 showing lower expression in cancer tissues compared with normal tissues maybe still play an important role in tumor proliferation.

KRAS and BRAF mutations in serum exosomes from patients with colorectal cancer in a Chinese population.

The efficacy of epidermal growth factor receptor- targeted therapy is significantly associated with Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-raf serine/threonine kinase proto-oncogene (BRAF) mutation in patients with colorectal cancer (CRC), for which the standard gene testing is currently performed using tumor tissue DNA. The aim of the present study was to compare the presence of KRAS and BRAF mutations in the serum exosome and primary tumor tissue from patients with CRC. Genomic DNA were extracted from the tumor tissues of 35 patients with histologically-confirmed CRC and exosomal mRNA were obtained from peripheral blood, which were collected from the corresponding patients prior to surgery. Three mutations in the KRAS gene (codons 12, 13 and 61) and a mutation in the BRAF gene (codon 600) were detected using a polymerase chain reaction-based sequencing method and their presence were compared between tumor tissues and the matched serum exosomes. The KRAS mutation rates in tumor tissues and the matched serum exosomes were 57.6 and 42.4%, respectively, which was not significantly different (P=0.063). The detection rate of the BRAF mutation was 24.2 and 18.2% in tumor tissues and the matched serum exosomes, respectively, and there was no significant difference (P=0.500). The patients with CRC that had a KRAS mutation of codon 12 in exon 2 in their tumor tissues and serum exosomes were significantly older compared with those without this mutation (tumor tissue, P=0.002; serum exosome, P=0.022). The sensitivity of KRAS and BRAF mutation detection using exosomal mRNA was 73.7 and 75%, respectively. The specificity of the detected mutations exhibited an efficiency of 100%, and the total consistency rate was 94.9 and 93.9% for KRAS and BRAF mutations, respectively. These results suggested that serum exosomal mRNA may be used as a novel source for the rapid and non-invasive genotyping of patients with CRC.

Low-dose tacrolimus combined with donor-derived mesenchymal stem cells after renal transplantation: a prospective, non-randomized study.

Calcineurin inhibitors, including tacrolimus, are largely responsible for advances in allotransplantation. However, the nephrotoxicity associated with these immunosuppressants impairs patients' long-term survival after renal allograft. Therefore, novel regimens that minimize or even eliminate calcineurin inhibitors could improve transplantation outcomes. In this pilot study, we investigated the use of low-dose tacrolimus in combination with mesenchymal stem cells (MSCs), which are immunosuppressive and prolong allograft survival in experimental organ transplant models. Donor-derived, bone marrow MSCs combined with a sparing dose of tacrolimus (0.04-0.05 mg/kg/day) were administered to 16 de novo living-related kidney transplant recipients; 16 other patients received a standard dose of tacrolimus (0.07-0.08 mg/kg/day). The safety of MSC infusion, acute rejection, graft function, graft survival, and patient survival were evaluated over ≥24 months following kidney transplantation. All patients survived and had stable renal function at the 24 month follow-up. The combination of low-dose tacrolimus and MSCs was as effective as standard dose tacrolimus in maintaining graft survival at least 2 years after transplantation. In addition, both groups had similar urea, urine protein, urinary RBC, urinary WBC, 24-h urine protein, and creatinine clearance rates from 7 days to 24 months after transplantation. Furthermore, no differences in the proportion of lymphocytes, CD19, CD3, CD34, CD38, and natural killer cells were detected between the control and experimental groups. None of the MSC recipients experienced immediate or long-term toxicity from the treatment. This preliminary data suggests that the addition of MSCs permits the use of lower dosages of nephrotoxic calcineurin inhibitors following renal transplantation.

The clinical feasibility and performance of an orthogonal X-ray imaging system for image-guided radiotherapy in nasopharyngeal cancer patients: Comparison with cone-beam CT.

The demand for greater accuracy of intensity-modulated radiotherapy (IMRT) has driven the development of more advanced verification systems for image-guided radiotherapy (IGRT). The purpose of this study is to investigate setup discrepancies measured between an orthogonal X-ray guidance system (XGS-10) and cone-beam computed tomography (CBCT) of Varian in the IMRT of patients with nasopharyngeal cancer (NPC). The setup errors measured by XGS-10 and CBCT at the treatment unit with respect to the planning CTs were recorded for 30 patients with NPC. The differences in residual setup errors between XGS-10 system and CBCT were computed and quantitatively analyzed. The time of image acquisition and image registration was recorded. The radiation doses delivered by CBCT and XGS-10 were measured using PTW0.6CC ionization chambers and a water phantom. The differences between setup errors measured by the XGS-10 system and CBCT were generally <1.5 mm for translations, indicating a reasonably good agreement between the two systems for patients with NPC in the translation directions of A-P (P = 0.856), L-R (P = 0.856) and S-I (P = 0.765). Moreover, compared with CBCT, XGS-10 took much shorter image acquisition and registration time (P <0.001) and delivered only a small fraction of extra radiation dose to the patients (P <0.001). These results indicate that XGS-10 offers high localization accuracy similar to CBCT and additional benefits including prompt imaging process, low imaging radiation exposure, real time monitoring, which therefore represents a potential attractive alternative to CBCT for clinical use.

Microarray analysis of the aberrant microRNA expression pattern in gliomas of different grades.

Previous studies have focused on miRNA expression in brain gliomas. However, both the expression pattern of miRNAs in gliomas of different grades and various miRNAs involved in malignant progression of gliomas are poorly understood. In the present study, we used miRNA microarray-based screening to investigate the miRNA expression profile in gliomas, which was further verified by qRT-PCR in selected miRNAs. In total, we found 13 differentially expressed miRNAs between gliomas and their matched surrounding tissues. Among them, 12 miRNAs were upregulated and only one (miR-4489) was downregulated compared with the control. Furthermore, the lower expression level of miR-4489 was confirmed by qRT-PCR in 26 glioma samples. Our microarray result revealed 8, 9 and 15 aberrantly expressed miRNAs in gliomas of World Health Organization (WHO) grade II-IV, respectively. Gene Ontology (GO) and Pathway analysis indicated that target genes of the 13 miRNAs were significantly enriched in central nervous system- and tumor-related biological processes and signaling pathways. The dysregulated miRNAs identified in the present study contribute to the tumorigenesis and malignant progression of gliomas and may serve as useful markers for advanced glioma pathological grading and prognosis.

Effect of CGRP-adenoviral vector transduction on the osteoblastic differentiation of rat adipose-derived stem cells.

Calcitonin gene-related peptide (CGRP) promotes osteoblast recruitment and osteogenic activity. However, no evidence suggests that CGRP could affect the differentiation of stem cells toward osteoblasts. In this study, we genetically modified adipose-derived stem cells (ADSCs) by introducing the CGRP gene through adenoviral vector transduction and investigated on cellular proliferation and osteoblast differentiation in vitro and osteogenesis in vivo as well. For the in vitro analyses, rat ADSCs were transducted with adenoviral vectors containing the CGRP gene (Ad-CGRP) and were cultured in complete osteoblastic medium. The morphology, proliferative capacity, and formation of localized regions of mineralization in the cells were evaluated. The expression of alkaline phosphatase (ALP) and special markers of osteoblasts, such as Collagen I, Osteocalcin (BPG) and Osteopontin (OPN), were measured by cytochemistry, MMT, RT-PCR, and Western blot. For the in vivo analyses, the Ad-CGRP-ADSCs/Beta-tricalcium phosphate (ß-TCP) constructs were implanted in rat radial bone defects for 12 weeks. Radiography and histomorphology evaluations were carried out on 4 weeks and 12 weeks. Our analyses indicated that heterogeneous spindle-shaped cells and localized regions of mineralization were formed in the CGRP-transduced ADSCs (the transduced group). A higher level of cellular proliferation, a high expression level of ALP on days 7 and 14 (p<0.05), and increased expression levels of Collagen I, BPG and OPN presented in transduced group (p<0.05). The efficiency of new bone formation was dramatically enhanced in vivo in Ad-CGRP-ADSCs/ß-TCP group but not in ß-TCP group and ADSCs/ß-TCP group. Our results reveal that ADSCs transduced with an Ad-CGRP vector have stronger potential to differentiate into osteoblasts in vitro and are able to regenerate a promising new tissue engineering bone in vivo. Our findings suggest that CGRP-transduced ADSCs may serve as seed cells for bone tissue engineering and provide a potential way for treating bone defects.

Activation of the phosphorylation of ATM contributes to radioresistance of glioma stem cells.

Ionizing radiation (IR) is currently the most efficient therapy available for malignant glioma. Unfortunately, this strategy is palliative due to the characteristics of radioresistance of malignant glioma. The aim of our study was to compare glioma stem cells (GSCs) with glioma cells (GCs) to determine whether GSCs are responsible for the radioresistance phenotype and to elucidate whether cell cycle checkpoint proteins are responsible for the radioresistance of GSCs. In this study, CD133 (a marker of brain cancer stem cells) and nestin were co-expressed in GSCs isolated from GCs. The percent of CD133+ cells in GSCs and GCs were >80 and <2%, respectively. Significantly more GSCs survived following 2, 4, 6 and 8 Gy IR than GCs. IR kills cancer cells primarily through DNA double-strand breaks (DSBs). The neutral comet assay is often used to intuitively show the level of DSBs. Significantly fewer GSCs showed DNA damage than GCs following 2 Gy IR. This demonstrated that GSCs are more resistant to in vitro radiation than GCs. Furthermore, activated ataxia telangiectasia mutated (ATM) is essential for the activation of downstream effector kinases, such as checkpoint kinase 2 (Chk2) and p53 which mainly contribute to the proper regulation of IR-induced arrest in the G1 phase. DNA damage induced by IR potently initiated activation of phosphorylation of the ATM, p53 and Chk2 checkpoint proteins. Activation of the phosphorylation of these checkpoint proteins was significantly higher in the GSCs compared to GCs. We found that inhibition of ATM activation induced cell cycle checkpoint defects and increased the rate of apoptosis of GSCs following IR. Our results suggest that GSCs were more resistant to radiation compared to GCs due to high expression of phosphorylated cell cycle checkpoint proteins, and inhibition of ATM could significantly reduce the radioresistance of GSCs and GCs. ATM may represent a source of radioresistance in GSCs and a target of improved radiosensitivity of GSCs.

[The mid-term follow-up of Coflex non-fusion internal fixation in the treatment of degenerative lumbar disease].

OBJECTIVE: To summarize the mid-term effectiveness of Coflex non-fusion internal fixation treatment of degenerative lumbar disease. METHODS: From October 2008 to December 2010, a retrospective analysis was carried out on 39 patients (29 males and 10 females) diagnosed as degenerative lumbar disease and treated with Coflex interspinous dynamic device, who had been followed up for 1 year at least, the average age was 45.5 years (range, 23 - 67 years). The results were assessed by Japanese Orthopedic Association (JOA) scores, visual analogue scale (VAS) scores, Oswestry disability index (ODI) scores and SF-36 scores; and the range of mobility (ROM), intervertebral disc height of the responsible and adjacent segments were measured on X-film before the operation and at last follow-up. Observed the therapeutic effect of the patients and compared the effect on the patients of different body mass index (BMI) and different age by the One-way analysis of variance and paired t test. RESULTS: The 39 patients were followed up for 30.9 months (range, 12 - 37 months). At the last follow-up, JOA, ODI, VAS and SF-36 scores were improved by 70% ± 12%, 54% ± 12%, 77% ± 10% and 51% ± 9%, and were statistically significant (t = -33.289, 26.448, 26.596 and -20.772, P = 0.00). Patients with BMI ≥ 25 kg/m(2) had lower improvement rates in the scores than those with BMI < 25 kg/m(2) (F = 10.561, 5.850, 5.651 and 6.519, P < 0.05). The patients were 50 years older or younger couldn't affected the improvement rates in the scores statistically (P > 0.05). There were no significant difference in remaining disc height (P > 0.05), except that the intervertebral disc height of L4-5 increased slightly compared with the preoperative (t = -2.819, P = 0.008). In addition to the ROM of L3-4, L5-S1 and L1-S1 were not significantly different from the preoperative(P > 0.05), the ROM of L4-5 were decreased (t = 12.598, P = 0.000). CONCLUSIONS: The mid-term effectiveness of Coflex non-fusion interspinous fixation in treatment of degenerative lumbar disease is worthy of recognition, and Coflex combined with Isobar has advantages in the treatment of multi-segment degenerative lumbar disease.

Differentiation of GFP-Bcl-2-engineered mesenchymal stem cells towards a nucleus pulposus-like phenotype under hypoxia in vitro.

Differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into a nucleus pulposus-like phenotype under hypoxia has been proposed as a potential therapeutic approach for intervertebral disc degeneration. However, limited cell viability under hypoxic conditions has restricted MSC differentiation capacity and thus restricted its clinical application. In this study, we genetically modified MSCs with an anti-apoptotic GFP-Bcl-2 gene and evaluated cell survival and functional improvement under hypoxia in vitro. Rat bone marrow MSCs were transfected by lentiviral vectors with the GFP-Bcl-2 gene (GFP-Bcl-2-MSCs). Cell proliferation and apoptosis were assessed, and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate phenotypic and biosynthetic activities. In addition, Alcian blue staining was used to detect the formation of sulfated glycosaminoglycans (GAGs) in the differentiated cells. We found that the Bcl-2 gene protected MSCs against apoptosis. We also observed that Bcl-2 over-expression reduced apoptosis by 40.61% in non-transfected MSCs and 38.43% in vector-MSCs to 18.33% in Bcl-2-MSCs. At 3days, the number of viable Bcl-2-MSCs was approximately two times higher than the number of MSCs or vector-MSCs under hypoxic conditions. RT-PCR showed higher expression of chondrocyte-related genes (Sox-9, aggrecan and type II collagen) in GFP-Bcl-2-MSCs cultured under hypoxia. The accumulation of proteoglycans in the pellet was 86% higher in GFP-Bcl-2-MSCs than in the control groups. Furthermore, the ratio of proteoglycans/collagen II in GFP-Bcl-2-MSCs was 6.2-fold higher compared to the MSC and vector-MSC groups, which denoted a nucleus pulposus-like differentiation phenotype. Our findings support the hypothesis that anti-apoptotic gene-modified MSCs can differentiate into cells with a nucleus pulposus-like phenotype in vitro, which may have value for the regeneration of intervertebral discs using cell transplantation therapy.

Twin pregnancy and partial hydatidiform mole following in vitro fertilization and embryos transfer: a novel case of placental mosaicism.

Twin pregnancy with mosaic partial hydatidiform mole (PHM) and survival of two healthy fetuses following in vitro fertilization and embryos transfer (IVF-ET) is a rare situation and is considered a challenge for management. A 32-year-old Chinese woman conceived twin pregnancy following IVF-ET. At 22 weeks' gestation, an additional intrauterine echogenic mass with features of PHM were shown by successive ultrasound examinations. At 35 weeks' gestation, two live male infants and two placentas were delivered by caesarean section (CS). Histologic examination of the abnormal placenta confirmed mosaic PHM. Genetic study showed the abnormal placental mosaicism (expressed in molar-69XXY and normal vili-46XY), co-existing with a hypospadia new-born (46XY) in one amniotic sac. However, the other one was normal. Serial serum ß-hCG levels showed a declining trend and serum ß-human chorionic gonadotropin (hCG) were undetectable at 6 months after delivery. The case demonstrated that it is possible to prolonged gestation by PHM under close surveillance during the entire pregnancy.