[Design of three-dimensional foraminal guide device and application in percutaneous endoscopic lumbar discectomy].

OBJECTIVE: To explore clinical application of the new three-dimensional foramen guide in percutaneous endoscopic lumbar discectomy. METHODS: Based on the principle of reverse positioning, a new three-dimensional foramen guide was designed. From May 2016 to May 2018, totally 40 patients with segmental lumbar disc herniation were underwent percutaneous endoscopic lumbar discectomy. The patients were divided into guide and control group, and 20 patients in each group. In guide group, there were 9 males and 11 females with an average age of (46.0±11.0) years old;5 patients on L3,4, 15 patients on L4,5; BMI was (25.4±3.2) kg /m2;three dimensional foramen guide was used to assist the operation. While in control group, there were 10 males and 10 females with an average age of (51.8±9.8) years old;4 patients on L3,4, 16 patients on L4,5;BMI was (24.8±3.5) kg /m2;the operation was completed with bare hands according to the experience. The puncture time, times of fluoroscopy and puncture between two groups were compared, as well as the preoperative and postoperative visual analogue scale (VAS) score and Japanese Orthopaedic Association (JOA) were compared. RESULTS: All patients had no serious complications, and successfully completed operation. Operation time, the times of fluoroscopy and puncture in guide group were better than those of control group (P<0.05). VAS score and JOA score between two groups were significantly relieved after operation (P<0.05), but there was no significant difference between two groups (P>0.05). CONCLUSION: The three dimensional foramen guide is compact in structure, simple in operation, which could reduce the time of puncture and damage of radiation, shorten the learning curve of puncture for beginners, and has certain clinical feasibility.

The integration of pore size and porosity distribution on Ti-6A1-4V scaffolds by 3D printing in the modulation of osteo-differentation.

PURPOSE: In this study, pore size and porosity distribution of porous Ti-6Al-4V scaffolds (pTi) were controlled by 3D printing. The effects of pore size distribution at a constant porosity, or porosity distribution at a constant pore size pertaining to functions of adhesion, proliferation, and differentiation of the mouse embryonic osteoblast precursor (MC3T3-E1) cells were researched separately. METHODS: 3D printing was used to design five groups of pTi, designated as PS300/HP, PS300/LP, PS500/HP, PS500/LP, and PS800/HP based on pore size and porosity distribution. MC3T3-E1 cells were cultured on pTi, and non-porous Ti-6Al-4V samples (npTi) were prepared as control. The pTi was characterized with the scanning electron microscopy (SEM). MC3T3-E1 cells were stained via AlamarBlue assay and viability and proliferation analyzed. The mRNA levels of alkaline phosphatase (ALP), osteocalcin (OCN), collagentype-1 (Col-1), and runt-related transcription factor 2 (Runx2) in MC3T3-E1 cells were analyzed by real-time PCR analysis. RESULTS: The average pore size and porosity of pTi were recorded as (301 ± 9 µm, 58.8 ± 1.8%), (300 ± 9 µm, 43.4 ± 1.3%), (501 ± 11 µm, 58.3 ± 1.2%), (499 ± 12 µm, 42.7 ± 1.1%), and (804 ± 10 µm, 58.9 ± 1.3%), respectively. SEM images confirmed active attachment of cells and oriented with the direction of metal rod after pTi/MC3T3-E1 co-culture for 3 and 7 days. In addition, MC3T3-E1 cells grown on the PS800/HP displayed significantly higher proliferation compared with each group after 3 days incubation (p < 0.05). Moreover, cells showed some degree of proliferation in all groups, with the highest value recorded for PS800/HP after culture for 7 days (p < 0.05). The gene expression pattern of ALP, OCN, Col-1, and Runx2 confirmed that these were down-regulated when pore size increased or porosity decreased of pTi (p < 0.05). CONCLUSION: The pTi facilitated the adhesion and differentiation of osteoblast when pore size decreased or porosity increased. The scaffold model resembles physical modification with porous structures, which has potential application in the surface modifications of Ti implant.

Intra-articular, single-shot co-injection of hyaluronic acid and corticosteroids in knee osteoarthritis: A randomized controlled trial.

The aim of the present study was to investigate whether the co-injection of hyaluronic acid (HA) and corticosteroids (CS) was superior to HA alone in the treatment of knee OA. A total of 120 participants with symptomatic knee OA were recruited and formed the intention-to-treat population for a 6-month follow-up. In the HA group, patients received a single-shot injection of 4 ml HA. In the HA&CS group, patients received a co-injection of 3 ml compound betamethasone solution and 4 ml HA. Visual analog scale (VAS), Western Ontario and McMaster University Osteoarthritis Index (WOMAC) and knee flexion motion were assessed as primary outcomes. Patients in the HA&CS group exhibited better pain relief and knee function at the time points of week 1, month 1 and month 3 (P<0.05). For the last follow-up at month 6, the values did not differ significantly between these two groups. Patients in both groups exhibited improvement in pain, knee function, and range of motion following injection. For the final follow-up at month 6, the mean VAS score, WOMAC score and knee flexion motion were still superior to that prior to treatment, but the values did not differ significantly. The co-injection of HA and CS provided a rapid improvement in pain relief, knee function, and range of motion, but did not differ significantly from that of HA alone in the long term effect.

Human Umbilical Cord-Derived Mesenchymal Stromal Cells Improve Left Ventricular Function, Perfusion, and Remodeling in a Porcine Model of Chronic Myocardial Ischemia.

UNLABELLED: : Stem cell therapy has emerged as a new strategy for treatment of ischemic heart disease. Although umbilical cord-derived mesenchymal stromal cells (UC-MSCs) have been used preferentially in the acute ischemia model, data for the chronic ischemia model are lacking. In this study, we investigated the effect of UC-MSCs originated from Wharton's jelly in the treatment of chronic myocardial ischemia in a porcine model induced by ameroid constrictor. Four weeks after ameroid constrictor placement, the surviving animals were divided randomly into two groups to undergo saline injection (n = 6) or UC-MSC transplantation (n = 6) through the left main coronary artery. Two additional intravenous administrations of UC-MSCs were performed in the following 2 weeks to enhance therapeutic effect. Cardiac function and perfusion were examined just before and at 4 weeks after intracoronary transplantation. The results showed that pigs with UC-MSC transplantation exhibited significantly greater left ventricular ejection fraction compared with control animals (61.3% ± 1.3% vs. 50.3% ± 2.0%, p < .05). The systolic thickening fraction in the infarcted left ventricular wall was also improved (41.2% ± 3.3% vs. 46.2% ± 2.3%, p < .01). Additionally, the administration of UC-MSCs promoted collateral development and myocardial perfusion. The indices of fibrosis and apoptosis were also significantly reduced. Immunofluorescence staining showed clusters of CM-DiI-labeled cells in the border zone, some of which expressed von Willebrand factor. These results suggest that UC-MSC treatment improves left ventricular function, perfusion, and remodeling in a porcine model with chronic myocardial ischemia. SIGNIFICANCE: Ischemic heart disease is the leading cause of death worldwide. Many patients with chronic myocardial ischemia are not suitable for surgery and have no effective drug treatment; they are called "no-option" patients. This study finds that umbilical cord-derived mesenchymal stromal cells transplanted by intracoronary delivery combined with two intravenous administrations was safe and could significantly improve left ventricular function, perfusion, and remodeling in a large-animal model of chronic myocardial ischemia, which provides a new choice for the no-option patients. In addition, this study used clinical-grade mesenchymal stem cells with delivery and assessment methods commonly used clinically to facilitate further clinical transformation.

[Inhibitory effects of human umbilical cord-derived mesenchymal stem cells on proliferation of peripheral blood mononuclear cells from spondyloarthritis patients].

OBJECTIVE: To explore the inhibitory effects of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on the proliferation of peripheral blood mononuclear cells (PBMC) from spondyloarthritis (SpA) patients. METHODS: A total of 12 SpA patients at Chinese PLA General Hospital were recruited from May 2012 to October 2012. Information on demographic characteristics, disease and functional activity was collected. Isolated PBMC were stimulated by phytohemagglutinin (PHA, 1 µg/ml) in the presence or absence of hUCMSC.The proliferation of hUCMSC was suppressed by irradiation with Co60 (30 Gy) before co-culturing with PBMC. The proliferation of PBMC was determined by Cell Counting Kit-8 (CCK-8). Cell cycle profiles of PBMC were analyzed by flow cytometry. The association of inhibitory effect of hUCMSC with the disease and functional activity of SpA patients was examined. RESULTS: After coculturing with hUCMSC by cell-to-cell contact for 5 days, the proliferation of PBMC stimulated by PHA (1 µg/ml) was significantly inhibited by hUCMSC in a dose-dependent manner.The inhibition rate of the proliferation of PBMC cocultured with hUCMSC by cell-to-cell contact was higher than that by Transwell culture (57% ± 17% vs 32% ± 12%, P < 0.01). Compared to PBMC cultured alone, a larger number of PBMC cocultured with hUCMSC were in phase G1 (86% ± 3% vs 68% ± 5%, P < 0.01) while a lower number of cells in phases S and G2 (8% ± 3% vs 26% ± 5%, P < 0.01). No association was found between the inhibitory effect of hUCMSC and the disease and functional activity. CONCLUSION: The proliferation of PBMC from SpA patients may be inhibited by hUCMSC. And hUCMSC have therapeutic potentials for SpA patients.

Treatment responses of procaterol and CD38 inhibitors in an ozone-induced airway hyperresponsiveness mice model.

Airway hyperresponsiveness (AHR) and airway inflammation are key pathophysiological features of many respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). To evaluate the treatment responses of procaterol and CD38 inhibitors in an ozone-induced AHR mice model, we hypothesized that procaterol and two synthetic CD38 inhibitors (Compounds T and H) might have therapeutic effects on the ozone-induced AHR mice model, and the nuclear factor-kappaB (NF-κB) pathway and the CD38 enzymatic activity might be involved in the mechanisms. With the exception of the Control group, ozone exposure was used to establish an AHR model. Male Kunming mice in the Procaterol and CD38 inhibitors groups were treated with an emulsifier of procaterol hydrochloride, Compound T or H. Results indicated that (1) no drug showed severe toxicity in this study; (2) ozone exposure induced airway inflammation and AHR; (3) intragastric treatment with procaterol and Compound T achieved potent therapeutic effects, but Compound H did not show any therapeutic effect; (4) the NF-κB pathway was involved in both the pathogenic mechanisms of ozone and therapeutic mechanisms of procaterol and Compound T; (5) however, the in vivo effect of Compound T was not caused by its inhibitory activity on CD38. Taken together, procaterol and Compound T are potentially good drugs to treat asthma and COPD complicated with ozone exposure.

[Inhibitory effect of human umbilical cord-derived mesenchymal stem cells on interleukin-17 production in peripheral blood T cells from spondyloarthritis patients].

In this study, the inhibitory effect of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on interleukin-17 (IL-17) production in peripheral blood T cells from patients with spondyloarthritis (SpA) were investigated, in order to explore the therapeutic potential of hUCMSC in the SpA. Peripheral blood mononuclear cells (PBMNC) were isolated from patients with SpA (n = 12) and healthy subjects (n = 6). PBMNC were cultured in vitro with hUCMSC or alone. The expression of IL-17 in CD4(+) T cells or γ/δ T cells were determined in each subject group by flow cytometry. IL-17 concentrations in PBMNC culture supernatants were measured by ELISA. The results indicated that the proportion of IL-17-producing CD4(+) T cells and IL-17-producing γ/δ T cells of SpA patients were 4.5 folds and 5 folds of healthy controls [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (0.75 ± 0.25)%, P < 0.01; CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.06 ± 0.02)%, P < 0.01]. After co-culture of PBMNC in patients with hUCMSC, the increased proportions of CD3(+)CD4(+)IL-17(+) cells and CD3(+)γδTCR(+)IL-17(+) cells in SpA patients were inhibited significantly by hUCMSC [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (1.81 ± 0.59)% (P < 0.01); CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.16 ± 0.06)% (P < 0.01]. In response to phytohemagglutinin (PHA, 1 µg/ml), PBMNC from SpA patients secreted more IL-17 than that from healthy control [(573.95 ± 171.68) pg/ml vs (115.53 ± 40.41) pg/ml (P < 0.01)]. In the presence of hUCMSC, PBMNC of SpA patients produced less amount of IL-17 [(573.95 ± 171.68) pg/ml vs (443.20 ± 147.94) pg/ml, (P < 0.01)]. It is concluded that the IL-17 production in peripheral blood T cells from SpA patients can be inhibited by hUCMSC, which have therapeutic potential for SpA.

Correlational study of the serum levels of the glial fibrillary acidic protein and neurofilament proteins in Parkinson's disease patients.

OBJECTIVE: To investigate changes in the serum levels of the glial fibrillary acidic protein (GFAP) and neurofilament proteins (NFs) in patients with Parkinson's disease (PD) and to determine their clinical significance. METHODS: In this study, 82 subjects were divided into 3 groups: the PD group, the acute cerebral infarction (ACI) group, and a normal control group. The serum levels of GFAP and NFs were measured using a sandwich ELISA assay. RESULTS: The serum levels of GFAP and NFs were significantly higher in the PD and the ACI groups than in the normal control group (P<0.05). There was no significant difference between the PD group and the ACI group (P>0.05). The serum level of GFAP in the PD group had no significant correlation with duration of the disease or age (P>0.05). The serum level of NFs in the PD group was significantly correlated with duration of the disease and age (P<0.05). CONCLUSIONS: The serum levels of GFAP and NFs were significantly higher in the PD group than in the normal group, indicating that astrocytic activity may remain elevated during the axonal degeneration that occurs over duration of the disease, although this activity is not specific to the disease.

[Optimization of in vitro culture conditions for human amniotic epithelial cells and expression of stem cell markers].

This study was purposed to optimize the culture conditions of the human amniotic epithelium cells (hAEC) in vitro, and detect the expression of hAEC pluripotent markers. Amnion tissues were separated from the underlying chorion through the spongy layer immediately after elective cesarean section of healthy pregnancy women at term. After the subsequent exposure to trypsin digestion, hAEC were cultured in DMEM with different supplements. The growth and proliferation potential of hAEC was evaluated, and the expression of cultured hAEC pluripotent markers was detected by using flow cytometry and immunohistochemistry methods. The results indicated that when being cultured in the mediums similar to that of embryonic stem cell culture supplemented with 10 ng/ml EGF, the hAEC grew better and the time for passage was shortened. In addition, compared to other culture conditions, under this condition, the cells could be passaged up to 5 times as much without obvious morphological changes, and the pluripotent marker SSEA-4 was detected in the cultured cells by flow cytometry. Meanwhile, the detection of immunofluorescence showed the expression of vimentin in cultured hAEC was strengthened as compared with primary cells. It is concluded that the culture condition similar to that for embryonic stem cells supplemented with EGF facilitates the proliferation and passage of hAEC in vitro.

[Down-regulation of Caspase-3 expression in precancerous lesions and its relation to gastric carcinogenesis].

OBJECTIVE: To study the level of expression of Caspase-3 protein in precancerous lesions of stomach and its relation to gastric carcinogenesis. METHODS: Formalin-fixed paraffin embedded tissues from 184 cases of gastric mucosa biopsy and surgically removed specimens, including gastric cancer (GC, N = 20), chronic atrophic gastritis (CAG, N = 6), atrophic gastritis with intestinal metaplasia (IM, N = 31), atrophic gastritis with dysplasia (DYS, N = 114) and normal controls (N = 13) were examined for expression of Caspase-3 protein and Ki-67 index by SABC immunohistochemistry, and for apoptosis by TdT-mediated dUTP biotin nick end labeling (TUNEL) method. Caspase-3, Ki-67 and TUNEL index were compared in different stages of gastric precancerous lesions and their correlation was analyzed. RESULTS: The positive index of Caspase-3 protein in severe DYS (29.8% +/- 3.9%) showed no significant difference compared with that in GC (26.9% +/- 3.0%, P > 0.05), but was significantly lower than that in low (58.3% +/- 4.2%) and moderate grade DYS (50.4% +/- 4.8%), CAG (68.3% +/- 3.3%) and IM (70.9% +/- 4.3%, P < 0.05). Caspase-3 positive index was significantly correlated with that of apoptosis detected by TUNEL (r = 0.94, P < 0.05). Ki-67 index in Caspase-3 protein positive group (18.3% +/- 2.2%) was significantly lower than that in Caspase-3 negative group (48.9% +/- 3.1%, P < 0.05). CONCLUSION: Caspase-3 protein expression was upregulated in CAG with or without IM and low or moderately low in DYS, while down-regulated in severe DYS and gastric carcinoma, and significantly positively correlated with cell apoptosis. It is suggested that down-regulated expression of Caspase-3 protein somehow contributes to gastric carcinogenesis through an imbalance between cell apoptosis and proliferation.

Loss of heterozygosity and microsatellite instabilities of fragile histidine triad gene in gastric carcinoma.

AIM: To detect the loss of heterozygosity (LOH) and microsatellite instabi1ities (MSI) of fragile histidine triad (FHIT) gene in gastric carcinoma and to study their association with the clinical pathological characteristics of gastric carcinoma. METHODS: LOH and MSI of FHIT gene were detected at four microsaterllite loci D3Sl3H, D3S4l03, D3Sl48l and D3S1234 using PCR in matched normal and cancerous tissues from 50 patients with primary gastric cancer. RESULTS: The average frequency of LOH and MSI of FHIT gene in gastric cancer was 32.4% and 26.4% respectively. LOH and MSI of FHIT gene in gastric cancer had no association with histological, Borrmann, and Lauren's classification. LOH of FHIT gene in gastric cancer was related to invasive depth. The frequency of FHIT LOH in gastric cancer with serosa-penetration was obviously higher than that in gastric cancer without serosa-penetration (73.5% vs 37.5%, P < 0.05). MSI of FHIT gene in gastric cancer was associated with the lymph node metastasis. The frequency of MSI in gastric cancer without lymph node metastasis was significantly higher than that in gastric cancer with lymph node metastasis (66.7% vs 34.3%, P < 0.05). CONCLUSION: LOH of FHIT gene is correlated with invasive depth of gastric carcinoma. MSI of FHIT gene is correlated with lymph node metastases. LOH and MSI of FHIT gene play an important role in carcinogenesis of gastric cancer.

[Frequencies distribution of human leukocyte antigen-B27 subtypes in healthy Chinese].

OBJECTIVE: To investigate the frequencies distribution of human leukocyte antigen (HLA)-B27 subtypes in unrelated healthy Chinese. METHOD: Polymerase chain reaction sequence-based typing (PCRSBT) was used to determine HLA high-resolution genotypes of 825 unrelated healthy Chinese. RESULTS: A total of 25 HLA-B27-positive individuals and 8 HLA-B27 subtypes were detected. These subtypes and their corresponding frequencies were B * 2704 (30.77%) , B * 2705 (23.08%), B * 2707 (19.23%), B * 2711 (7.69%), B * 2712 (7.69%), B * 2701 (3.85%), B * 2713 (3.85%) and B * 2721 (3.85%). CONCLUSION: The data obtained through PCR-SBT method may serve as important reference for the research of relationship between HLA-B27 subtypes and some diseases such as ankylosing spondylitis.

[HLA-A, B, DRB1 gene polymorphism of Beijing population was studied by high-resolution polymerase chain reaction sequence-based typing].

OBJECTIVE: To investigate the gene polymorphism of human leukocyte antigen (HLA)-A, B, DRB1 loci in the population of Beijing region, and research on the application feasibility of polymerase chain reaction sequence-based typing (PCR-SBT) method. METHODS: PCR-SBT method was applied to determine HLA- A, B, DRB1 genotypes of 618 unrelated healthy individuals of Beijing region. RESULTS: A total of 84 different alleles and 199 genotypes of HLA-A, 143 alleles and 366 genotypes of HLA-B, 122 alleles and 286 genotypes of HLA-DRB1 were detected. CONCLUSION: The results showed the characteristics of HLA-A, B, DRB1 distributions, and provided more comprehensive and accurate gene data that may serve as normal reference values for all of Beijing people.

[Mutation of mitochondrial 12S rRNA in gastric carcinomas and its significance].

OBJECTIVE: To detect the alterations of mitochondrial 12S rRNA in patients with gastric cancer, and further evaluate their effects on development of gastric carcinomas. METHODS: Mitochondrial 12S rRNA of 22 samples of gastric cancer tissues and 22 corresponding normal gastric mucosa taken from the distal portion of surgical specimens were PCR amplified, followed by direct DNA sequencing. Laser capture microdissection technique (LCM) was used to isolate cancerous cells and dysplastic cells from patients with specific mutations. Denaturing high-performance liquid chromatography (DHPLC) plus allele-specific PCR (AS-PCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE) were applied to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplastic cells. Finally, RNAdraw bio-soft was used to analyze the RNA secondary structure of mutant type 12S rRNA. RESULTS: Compared with mitomap database, some variations were firstly found, among which np652 G insertion and np716 T-G transversion were only found in cancers. There existed statistically significant difference in variant frequency of 12S rRNA between intestinal type and diffuse type of gastric carcinoma, 5/17 (29.4%) and 12/17 (70.6%) respectively (P < 0.05). DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutation. Variant frequency of 12S rRNA in cancer was higher than that in dysplasia (P < 0.01). 12S rRNA 652G insertion had more adverse effect on secondary structure stability of 12S rRNA than T-G transversion did. CONCLUSION: Highly variant frequency of mitochondrial 12S rRNA may be associated with intestinal type of gastric cancer. Most parts of variations exist in both cancer and normal tissues and may not be characteristic of tumor specificity. However np652 G insertion and np716 T-G transversion may possess some molecular significance on gastric cancerogenesis. During the process of progression from normality through dysplasia to cancer, 12S rRNA tended to transit from homoplasmy (wild type) and heteroplasmy to homoplasmy (mutant type, np717 T-G).

Loss of heterozygosity on 10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions.

AIM: To investigate the loss of heterozygosity (LOH) and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. METHODS: Thirty cases of normal gastric mucosa, advanced and early stage gastric cancer, intestinal metaplasia, atrophic gastritis, and atypical hyperplasia were analyzed for PTEN LOH and mutations within the entire coding region of PTEN gene by PCR-SSCP denaturing PAGE gel electrophoresis, and PTEN mutation was detected by PCR-SSCP sequencing followed by silver staining. RESULTS: LOH rate found in respectively atrophic gastritis was 10% (3/30), intestinal metaplasia 10% (3/30), atypical hyperplasia 13.3% (4/30), early stage gastric cancer 20% (6/30), and advanced stage gastric cancer 33.3% (9/30), None of the precancerous lesions and early stage gastric cancer showed PTEN mutations, but 10% (3/30) of the advanced stage gastric cancers, which were all positive for LOH, showed PTEN mutation. CONCLUSION: LOH of PTEN gene appears in precancerous lesions, and PTEN mutations are restricted to advanced gastric cancer, LOH and mutation of PTEN gene are closely related to the infiltration and metastasis of gastric cancer.

Preliminary DNA identification for the tsunami victims in Thailand.

The 2004 Southeast Asia Tsunami killed nearly 5,400 people in Southern Thailand, including foreign tourists and local residents. To recover DNA evidence as much as possible from the seriously decomposed bodies, we explored procedures of sample preparation from both bone and tooth samples as well as both mitochondrial and nuclear markers. Despite having failed to recover enough DNA for nuclear marker typing, we succeeded in obtaining fully informative results for mitochondrial markers (HV1 and HV2) from 258 tooth samples with a success rate of 51% (258/507). Using an organic DNA extraction method coupled with an ultrafiltration step, we obtained 16 STR (including 13 CODIS loci, one sex discrimination locus, and two Identifiler loci) profiles for 834 samples with a success rate of 79% (834/1,062). In addition, by comparing the allelic frequencies between the typed samples as a group and other index populations, we conclude that the Thai tsunami victims are a combined group of several populations. Our results provide valuable evidence and protocols for the future forensic practice.

Mutations of mitochondrial 12S rRNA in gastric carcinoma and their significance.

AIM: To detect the variations of mitochondrial 12S rRNA in patients with gastric carcinoma, and to study their significance and the relationship between these variations and the genesis of gastric carcinoma. METHODS: PCR amplified mitochondrial 12S rRNA of 44 samples including 22 from gastric carcinoma tissues and 22 from adjacent normal tissues, was detected by direct DNA sequencing. Then laser capture microdissection technique (LCM) was used to separate the cancerous cells and dysplasia cells with specific mutations. Denaturing high performance liquid chromatography (DHPLC) plus allele-specific PCR (AS-PCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE) were used to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplasia cells. Finally, RNAdraw biosoft was used to analyze the RNA secondary structure of mutant-type 12S rRNA. RESULTS: Compared with Mitomap database, some new variations were found, among which np652 G insertion and np716 T-G transversion were found only in cancerous tissues. There was a statistic difference in the frequency of 12S rRNA variation between intestinal type (12/17, 70.59%) and diffusive type (5/17, 29.41%) of gastric carcinoma (P<0.05). DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutations. The frequency of 12S rRNA variation in cancerous cells was higher than that in dysplasia cells (P<0.01). 12S rRNA np652 G insertion showed obviously negative effects on the stability of 12S rRNA secondary structure, while others such as T-G transversion did not. CONCLUSION: The mutations of mitochondrial 12S rRNA may be associated with the occurrence of intestinal-type gastric carcinoma. Most variations exist both in gastric carcinomas and in normal tissues, and they might not be the characteristics of tumors. However, np652 G insertion and np716 T-G transversion may possess some molecular significance in gastric carcinogenesis. During the process from normality to dysplasia, then to carcinoma, 12S rRNA tends to convert from homoplasmy (wild type) to heteroplasmy, then to homoplasmy (mutant type, np717 T-G).

Pathobiological significance of vascular endothelial growth factor and Maspin expressions in human gastric carcinoma.

AIM: To investigate the correlation between expression of vascular endothelial growth factor (VEGF) and cell differentiation, invasion, metastasis and Maspin expression in gastric carcinoma. METHODS: Formalin-fixed paraffin-embedded tissue specimens from 73 cases of gastric carcinoma were studied with SP immunohistochemistry, using anti-VEGF monoclonal antibody, and thirty-nine of them were studied using anti-Maspin monoclonal antibody. VEGF expression was compared with the clinical stage, lymph node metastasis, and Borrmann's and WHO's classification of gastric carcinoma. RESULTS: The positive rate of VEGF expression was significantly higher in adjacent non-carcinoma epithelia (ANCE) than in non-metaplastic, non-carcinoma gastric epithelia (NMNCE), which were at least 4 cm distant from the primary tumor (P = 0.000, chi2 = 73.03). The positive rate of VEGF expression was significantly higher in advanced gastric carcinoma (AGC) than in early gastric carcinoma (EGC) (P = 0.032, chi2 = 4.62). The positive rate of VEGF expression in gastric carcinomas with lymph node metastases was significantly higher than that in those without metastasis (P = 0.006, chi2 = 7.47). Maspin was weakly expressed in 16 out of 39 cases of NMNCE, and the positive immunoreaction was limited to gland cells of the stomach body. There was no significant correlation between the expression of VEGF and histological or gross classifications, and correlation between the expressions of VEGF and Maspin in gastric carcinoma (P = 0.648, chi2 = 0.21). CONCLUSION: Expression of VEGF is significantly correlated to the malignant biological behaviors of gastric carcinoma, but there is no significant correlation between the expression of VEGF and Maspin.

Pathobiological behavior and molecular mechanism of signet ring cell carcinoma and mucinous adenocarcinoma of the stomach: a comparative study.

AIM: To elucidate the distinctive pathobiological behavior between signet ring cell carcinoma (SRC) and mucinous adenocarcinoma of the stomach. METHODS: Based on the histological growth patterns and cell-functional differentiation classifications of stomach carcinoma, we conducted a series of comparative studies. All paraffin-embedded and frozen blocks were collected from the files of Cancer Institute of China Medical University. On the basis of histopathological observation, we applied enzymatic and mucous histochemistry, immunohistochemistry, flow cytometry (FCM) and molecular biology to compare these two categories of gastric cancers in terms of the DNA ploidy, proliferative kinetics, the expression of gastric carcinoma associated gene product and instabilities of mitochondrial DNA (mtDNA). RESULTS: Gastric SRC was commonly seen in females below 45 years, mostly presenting diffuse growth and ovary or uterine cervix metastasis. The majority of SRC were absorptive and mucus-producing functional differentiation type (AMPFDT), which growth relied on estrogen. Meanwhile, stomach mucinous adenocarcinomas were mostly observed in males over 50 years, prone to massive growth or nest growth and extensive peritoneal infiltration, showing two categories of cell-functional differentiation types: AMPFDT and mucus-secreting functional differentiation type (MSFDT). Expressions of ER, enzyme c-PDE and 67kDaLN-R in SRC were evidently higher than that in mucinous adenocarcinoma, while expressions of LN, CN-IV, CD44v6, and PTEN protein were obviously lower in SRC than that in mucinous adenocarcinoma (P<0.05). There was no statistic significance in VEGF, ECD and instabilities of mtDNA (P>0.05) between the above two gastric carcinomas. CONCLUSION: Though SRC and mucinous adenocarcinoma were both characterized by abundant mucus-secretion, they were quite different in morphology, ultrastructure, cell-functional differentiation and protein expression, indicating different mechanisms of carcinogenesis. We concluded that combining histological growth patterns, cell-functional differentiation type with tumor related markers might be significant in early diagnosis and prognosis assessment for SRC and mucinous adenocarcinoma of the stomach.

Variations of mitochondrial D-loop region plus downstream gene 1 2S rRNA-tRNA(phe) and gastric carcinomas.

AIM: To explore the instabilities, polymorphisms and other variations of mitochondrial D-loop region and downstream gene 12S rRNA-tRNA(phe) in gastric cancers, and to study their relationship with gastric cancer. METHODS: Three adjacent regions (D-loop, tRNA(phe) and 12S rRNA) were detected for instabilities, polymorphisms and other variations via PCR amplification followed by direct DNA sequencing in 22 matched gastric cancerous tissues and para-cancerous normal tissues. RESULTS: PolyC or (CA) (n) instabilities were detected in 13/22(59.1 %) gastric cancers and 9/22(40.9 %) in the control (P>0.05). There existed 2/12(16.7 %) and 6/10(60 %) alterations of 12S rRNA-tRNA(phe) in well differentiated gastric cancers and poorly differentiated ones, respectively (P<0.05). Some new variations were found, among which np 318 and np 321 C-T transitions in D-loop region were two of the five bases for H-strand replication primer. np 523 AC-deletion and np 527 C-T transition occurred at mtTF1 binding site (mtTFBS), which were associated with the transcription of downstream mitochondrial genome. Seven samples showed the np 16 182 polyC instabilities, five of which simultaneously showed np 16 189 T-C transitions. CONCLUSION: There is no statistic significance of instabilities and polymorphisms in mitochondrial D-loop region between gastric cancerous and para-cancerous normal tissues, which suggests that the instability might relate to heredity or be dependent on aging. There is a significant correlation between differentiation degree of gastric cancer and variant frequencies of 12S rRNA-tRNA(phe). The poorly differentiated gastric cancers are more prone to 12S rRNA-tRNA(phe) variations, or gastric cancers with 12S rRNA-tRNA(phe) variations are more likely to be poorly differentiated. np 16 189 T-C transition may be one of the important reasons for polyC instability in gastric cancer.