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ABSTRACT Objective: To observe the therapeutic effect of gentiopicroside, as the main component of Gentianaceae, on wounds in pressure injury (PI) model rats and explore its mechanism. Method: Male Sprague Dawley rats were randomly divided into control group, model group and gentiopicroside groups (50, 100 and 200 mg·kg-1·d-1 for 9 consecutive days). The mice's skeletal muscle fibroblast line NOR-10 cells were collected after being treated with gentiopicroside (0.2~5.0 M) and basic fibroblast growth factor receptor 1 (bFGFR1) inhibitor (5.0 M SU5402) for 7 days. Results: Compared to the model group, the gentiopicroside groups showed significantly increased wound healing rates, reduced inflammatory cells in the wound tissues, and significantly increased expression levels of proliferating cell nuclear antigen (PCNA) and bFGFR1, accompanied by increased proliferation of new myofibroblasts. Gentiopicroside upregulated the mRNA expression of bFGFR1 and PCNA in NOR-10 cells in a dose-dependent manner; however, SU5402 reversed the effect of gentiopicroside. Conclusion: Gentiopicroside may promote myofibroblast proliferation by upregulating the expression of bFGFR1 and PCNA and ultimately accelerating the healing of PI wounds.
RESUMO Objetivo: Observar o efeito terapêutico do gentiopicrosídeo como principal componente das Gentianáceas em feridas de lesão por pressão (LP) em modelos de ratos e explorar seu mecanismo. Métodos: Ratos Sprague Dawley machos foram divididos aleatoriamente em grupo controle, grupo modelo e grupos gentiopicrosídeo (50, 100 e 200 mg·kg-1·d-1 por 9 dias consecutivos). As células NOR-10 da linha de fibroblastos do músculo esquelético de camundongos foram coletadas após serem tratadas com gentiopicrosídeo (0,2~5,0 μM) e inibidor do receptor 1 do fator de crescimento fibroblástico básico (bFGFR1) (5.0 μM SU5402) por 7 dias. Resultados: Em comparação com o grupo modelo, os grupos gentiopicrosídeo apresentaram taxas de cicatrização de feridas significativamente maiores, menos células inflamatórias nos tecidos da ferida e níveis de expressão de antígeno nuclear de proliferação celular (PCNA) e bFGFR1 significativamente maiores, acompanhados por aumento da proliferação de novos miofibroblastos. O gentiopicrosídeo regulou positivamente a expressão de mRNA de bFGFR1 e PCNA em células NOR-10 de maneira dependente da dose, enquanto o SU5402 reverteu o efeito do gentiopicrosídeo. Conclusão: O gentiopicrosídeo pode promover a proliferação de miofibroblastos, suprarregulando a expressão de bFGFR1 e PCNA e, em última análise, acelerando a cicatrização de feridas de LP.
RESUMEN Objetivo: Observar el efecto terapéutico del gentiopicrósido como componente principal de la Gentianaceae en heridas por lesión por presión (LP) en modelos de ratas y explorar su mecanismo. Método: Se dividieron aleatoriamente ratas macho Sprague Dawley en grupo control, grupo modelo y grupos gentiopicrósido (50, 100 y 200 mg·kg-1·d-1 durante 9 días consecutivos). Se recogieron células NOR-10 de la línea de fibroblastos de músculo esquelético de ratón después de ser tratadas con gentiopicrósido (0.2~5.0 μM) y un inhibidor del receptor 1 del factor de crecimiento de fibroblastos básico (bFGFR1) (5.0 μM SU5402) durante 7 días. Resultados: En comparación con el grupo modelo, los grupos de gentiopicrósido mostraron tasas de curación de heridas significativamente más altas, menos células inflamatorias en los tejidos de la herida y niveles de expresión significativamente mayores del antígeno nuclear de proliferación celular (PCNA) y bFGFR1, acompañados de una mayor proliferación de nuevos miofibroblastos. El gentiopicrósido podría regular positivamente la expresión de ARNm de bFGFR1 y PCNA en células NOR-10 de manera dependiente de la dosis, sin embargo, SU5402 revirtió el efecto del gentiopicrósido. Conclusión: El gentiopicrósido puede promover la proliferación de miofibroblastos al aumentar la expresión de bFGFR1 y PCNA y, en última instancia, acelerar la cicatrización de las heridas de LP.
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Humanos , Cicatrização , Úlcera por Pressão , Antígeno Nuclear de Célula em Proliferação , Gentianaceae , Receptor Tipo 1 de Fator de Crescimento de FibroblastosRESUMO
Fibroblast growth factor receptor (FGFR) 2 gene fusion plays an important role in the pathogenesis of cholangiocarcinoma (CCA) . The method of targeting FGFR2 has been listed as the major therapy for advanced CCA. Adenosine triphosphate (ATP) -competitive FGFR inhibitors, represented by infigratinib and pemigatinib, effectively delay tumor progression and prolong patients survival, and are the first-line drugs for advanced CCA patients with FGFR2 fusion. However, almost all the patients treated with infigratinib eventually develop resistance, which require the combination with other drugs. Futibatinib may serve as a later-line drug for advanced CCA patients with V564F mutation after infigratinib resistance. For the infigratinib-resistant CCA patients harboring aberrant activation of the mitogen-activated protein kinase (MAPK) pathway, combination of the MAPK inhibitor or the heat shock protein 90 inhibitor may be considered as a novel therapeutic option.
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Objective:To evaluate regulatory effects of fibroblast growth factor receptor 3 (FGFR3) and human papillomavirus type 2 (HPV2) E2 protein on the differentiation of an immortalized human keratinocyte line HaCaT and a normal human epidermal keratinocyte line NHEK.Methods:In both HaCaT and NHEK cells, HPV2 E2-stably transfected cell lines (HPV2 E2-transfected groups) were established by using the lentivirus transfection method, wide-type FGFR3-overexpressing cells (FGFR3-WT transfected groups) and FGFR3-K650E mutant-overexpressing cells (FGFR3-K650E transfected groups) were constructed by using the plasmid transfection method, and cells transfected with blank vectors served as control groups (blank vector control groups). Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of HPV2 E2, and Western blot analysis to determine the protein expression of HPV2 E2, FGFR3, and keratinocyte differentiation markers including loricrin, filaggrin, as well as involucrin. Laser scanning confocal microscopy was conducted to observe the spatial localization of HPV2 E2 and FGFR3 in HaCaT cells. Statistical analysis was carried out by using two-independent-sample t test for the comparison between two groups, one-way analysis of variance for the comparison among multiple groups, and Dunnett t-test for multiple comparisons. Results:The HPV2 E2-stably transfected cell lines were successfully constructed, and the expression of HPV2 E2 FLAG protein was significantly higher in the HPV2 E2-transfected groups than in the blank vector control groups in both HaCaT and NHEK cells ( t = 13.71, 25.91, respectively, both P < 0.001) ; both FGFR3-WT and FGFR3-K650E were successfully overexpressed in both HaCaT and NHEK cells, and the FGFR3 protein expression was significantly higher in the FGFR3-WT transfected groups and the FGFR3-K650E transfected groups than in the blank vector control groups ( F = 473.90, 579.90, respectively, both P < 0.001). In both HaCaT and NHEK cells, the expression of keratinocyte differentiation markers including loricrin, filaggrin, and involucrin was significantly upregulated in the HPV2 E2-transfected groups, the FGFR3-WT transfected groups, and the FGFR3-K650E transfected groups than in the blank vector control groups (all P < 0.05). In the HPV2 E2-stably transfected HaCaT and NHEK cells, the expression of loricrin, filaggrin, and involucrin was significantly down-regulated in the HPV2 E2 + FGFR3-WT transfected groups and the HPV2 E2 + FGFR3-K650E transfected groups than in the HPV2 E2 + blank vector groups (all P < 0.05). Laser scanning confocal microscopy showed the spatial co-localization of HPV2 E2 and FGFR3 in the nuclear membrane and cytoplasm of HaCaT cells. Conclusion:HPV2 E2 and FGFR3 could both induce the differentiation of HaCaT and NHEK cells, while FGFR3 could inhibit HPV2 E2-induced differentiation trend of HaCaT and NHEK cells, which may be related to the cellular spatial co-localization of HPV2 E2 and FGFR3.
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A pedigree with familial acanthosis nigricans presenting with atypical clinical symptoms was reported. The 4-year-old female proband began to develop black patches on the neck and abdomen since the age of 1 year, which had gradually spread to the lips and front of the trunk in recent years. Reflectance confocal microscopy of the abdominal skin showed downward extension and twisting of dermal papillary rings with formation of gully-like structures, and moderately to highly refractive particles in the dermal papillary rings. The proband′s father and grandmother had similar medical history, but the pigmentation spontaneously subsided with age, leaving only local thickened skin lines. Peripheral blood samples were collected from the proband, her parents and grandmother, and panel-based targeted sequencing of peripheral blood DNA was performed for the proband. Sequencing showed a missense mutation c.1949A>C (p.Lys650Thr) in exon 14 of the FGFR3 gene in the proband, and Sanger sequencing confirmed the presence of this mutation in the proband and her father and grandmother. A diagnosis of familial acanthosis nigricans was made.
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ABSTRACT Objective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer. Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software. Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient. Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.
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Neoplasias da Bexiga Urinária/genética , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Brasil , RNA , Prevalência , Amarelo de Eosina-(YS) , Hematoxilina , Músculos/metabolismo , Músculos/patologia , MutaçãoRESUMO
Preoperative chemo- and radiotherapeutic strategies followed by surgery are currently a standard approach for treating locally advanced gastric and esophagogastric junction cancer in Western countries. However, in a large number of cases, the tumor is extremely resistant to these treatments and the patients are exposed to unnecessary toxicity and delayed surgical therapy. The current clinical trials evaluating the combination of preoperative systemic therapies with modern targeted and immunotherapeutic agents represent a unique opportunity for identifying predictive biomarkers of response to select patients that would benefit the most from these treatments. However, it is of utmost importance that these potential biomarkers are corroborated by extensive preclinical and translational research. The aim of this review article is to present the most promising biomarkers of response to classic chemotherapeutic, anti-HER2, antiangiogenic, and immunotherapeutic agents that can be potentially useful for personalized preoperative systemic therapies in gastric cancer patients.
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Humanos , Biomarcadores , Junção Esofagogástrica , Instabilidade de Microssatélites , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Neoplasias Gástricas , Pesquisa Translacional BiomédicaRESUMO
Background: Dysregulation of long non-coding RNAs (lncRNAs) has been found in a variety of malignancies. Previous study revealed that lncRNA-BBOX1-2 was up-regulated in gastric cancer and fibroblast growth factor receptor 1 (FGFR1) might be the potential target gene of lncRNA-BBOX1-2. Aims: To investigate the expressions and significance of lncRNA-BBOX1-2 and FGFR1 in gastric cancer. Methods: Expressions of lncRNA-BBOX1-2 and FGFR1 were detected by real-time PCR in 45 cases of gastric cancer tissues and the adjacent normal tissues. The correlations of these expressions with clinicopathological features of gastric cancer were analyzed. ROC curve analysis was used to assess the performance of lncRNA-BBOX1-2 and FGFR1 in distinguishing cancerous tissue and normal tissue and predicting lymph node metastasis. After transfected with siRNA-BBOX1-2, the expression of FGFR1 in human gastric cancer cell line SGC-7901 was detected by real-time PCR and Western blotting. Results: Both lncRNA-BBOX1-2 and FGFR1 were overexpressed in gastric cancer tissues than in adjacent normal tissues (P<0.05). The expression level of lncRNA-BBOX1-2 was positively correlated with lymph node metastasis and TNM stage (P<0.05), and the expression level of FGFR1 was positively correlated with tumor differentiation, depth of invasion, lymph node metastasis and TNM stage (P<0.05). The expression levels of lncRNA-BBOX1-2 and FGFR1 demonstrated a modest correlation with each other (r=0.344, P<0.05). When lncRNA-BBOX1-2 was knocked out by RNA interference, mRNA and protein expressions of FGFR1 were significantly decreased in SGC-7901 cells (P<0.05). ROC curve analysis showed that lncRNA-BBOX1-2 and FGFR1 might be potential biomarkers for initiation (AUC: 0.916 and 0.862, respectively) and lymph node metastasis (AUC: 0.720 and 0.774, respectively) of gastric cancer. Conclusions: LncRNA-BBOX1-2 and FGFR1 are up-regulated in gastric cancer tissues. LncRNA-BBOX1-2 might be a positive regulator of FGFR1 and their cooperation are involved in modulating tumorigenesis and development of gastric cancer.
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Objective: To observe the impact of vascular calcification on kidney injury rats with the expressions of β-Klotho, fibroblast growth factor receptor 1 (FGFR1) in kidney tissue in order to find the predictor for early chronic kidney disease (CKD), to provide the prevention and investigation basis of vascular calcification and CKD. Methods: Vascular calcification model was induced by vitamin D3 and nicotine injection in experimental rats and the animals were divided into 2 groups: Normal control group and Calcification group. n=6 in each group. Serum levels of creatinine and urea nitrogen were examined by sarcosine oxidase method and UV-glutamate dehydrogenase method respectively; blood levels of calcium and phosphorus were detected by biochemistry method; kidney tissue alkaline phosphatases (ALP) activity was measured by ALP detection kit, protein expressions of β-Klotho and FGFR1 were assessed by ELISA.Results: Compared with Normal control group, Calcification group showed increased serum levels of creatinine (35.200±4.087) umol/L vs (26.000±5.0990) umol/L and urea nitrogen (6.900±0.623) mmol/L vs (5.400±0.803) mmol/L, both P<0.05; elevated kidney tissue ALP activity (60.510±31.090) U/g vs (26.590±8.664) U/g and β-Klotho protein expression (9.052±1.238) ng/mg vs (6.860±1.036) ng/mg, both P<0.05. Blood levels of calcium, phosphorus and kidney tissue FGFR1 protein content were similar between 2 groups. Conclusion: Large dose vitamin D3 and nicotine injection may induce vascular calcification and early CKD symptom in experimental rats; β-Klotho protein expression was significantly increased suggesting that β-Klotho had been involved in the early regulation of vascular calcification and it could be used for the early diagnosis of CKD at certain point.
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Objective To explore the value of prenatal genetical diagnosis by mutation analysis of achondroplasia (ACH) fibroblast growth factor receptor 3 (FGFR3) gene.Methods Genomic DNA from nine ACH patients and their parents in Gansu Maternal and Child Health Hospital from July,2010 to December,2014 was prepared for polymerase chain reaction.Direct sequencing revealed the samples were performed after amplification of exon 10 of FGFR3 containing the potential mutation.Fetal DNA was extracted from cells in both amniotic fluid and umbilical cord,and then exon 10 of FGFR3 was also tested.Three fetuses with short-limb dysplasia were also included and prenatal diagnosis was offered to them through amniocentesis or cordocentesis.Results Prenatal ultrasonography test showed shorter femoral length,which was less than 2-3 standard deviation of normal reference dysplasia fetal performance for femoral short.Femur length is lower than 2-3 standard deviation minus normal value,and discrepancy in biparietal diameter compared with fetuses at the same gestational age.In the four families with one ACH parent,c.1138G > A heterozygous mutation was detected in all of the four mothers,while two fetuses among them showed c.1138G > A heterozygous mutation mutation and the other two were normal.There were other two fetuses with c.1138G > A heterozygous mutation from other two families,one's father had c.1138G > A heterozygous mutations,but not the mother,the other had c.1138G > A heterozygous mutations in both the mother and father.Among the three families with unaffected parents but each had a de novo c.1138G > A mutation child,no mutation of c.1138G > A genotype was detected in their fetuses,neither in the three fetus with short limb dysplasia.Four fetuses with a c.1138G > A mutation and three with short-limb dysplasia were terminated.The other five fetuses whose genotype was normal were born and healthy with normal phenotype at one-year-old follow-up.Conclusion FGFR3 genetic analysis could provide information for genetic counseling and prenatal diagnosis for ACH parents or parents who had an ACH baby to prevent birth defect.
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Fibroblast growth factor receptor 3(FGFR3)plays important roles in cell proliferation,diffe rentiation,and angiogenesis.Recent studies have demonstrated that FGFR3 is associated with progression of breast cancer and has effects in endocrine therapy resistance breast cancer.It has also been showed that FGFR3 is correlated with breast cancer prognosis.
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Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.
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Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.
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Feminino , Humanos , Recém-Nascido , Gravidez , Blastômeros , Osso e Ossos , Nanismo , Estruturas Embrionárias , Feto , Deformidades Congênitas dos Membros , Lordose , Linfócitos , Biologia Molecular , Parto , Reação em Cadeia da Polimerase , Diagnóstico Pré-Implantação , Prostaglandinas D , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Análise de SequênciaRESUMO
Objective To identify the genetic mechanism of fetuses with short limbs deformity.Methods From Aug.2008 to Aug.2011,ten fetuses with obvious short limbs were found in ultrasound screening performed at 18-24 and (or) 30-32 gestational weeks and underwent artificial induced labor with the patient' consent.Amniotic fluid or cord blood of the fetuses was collected for karyotyping analysis and detection of mutation point of fibroblast growth factor receptor 3 (FGFR3)gene by polymerase chain reaction and gene sequencing.One fetus (case 3) who presented with achondrogenesis underwent sequencing of SLC26A2 and Trip11 gene meanwhile.Results Among the 10 fetuses with short limbs deformity,five cases were found during second trimester and five during third trimester.Nine cases were identified as normal karyotype and one was chimera (46,XY/45,XY,- 18).One fetus carried a rare FGFR3 mutation of c.1108G>T (G370C) and was diagnosed as thanatophoric dysplasia at 21+3 weeks.Three fetus carried c.1138G>A (G380R) mutation and were diagnosed as achondroplasia.These four families had low recurrent risk because no gene mutations were found in the parents.Three mothers of these four fetuses were pregnant again and had normal neonates now.No mutations were found in all gene sequencing in case 3.Conclusions Karyotyping analysis and sequencing of FGFR3 gene could find causative gene mutations and provide genetic counselling and prenatal diagnosis for some fetuses with short limbs deformity.In the third trimester,achondroplasia is the most possible diagnosis when short limbs fetus is found by ultrasound.
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Objective:To explore the relationship between the polymorphisms in gene FGFR1, FGF10, FGFI8 and the nonsyndromic cleft lip with or without cleft palate (NS CLP) in Chinese population. Methods: Genomic DNA was isolated from peripheral lymphocytes of 75 patients with NS CLP and their parents and 75 unimpaired healthy children. The polymorphisms in FGFRI gene rs13317, p. E467K, p. M3691 and p. S393S, FGF10 gene rs1448037 and FGFI8 gene rs4043716 were detected by applying three-dimensional (3-D) polyacrylamide gel microarray technology. The data were performed using statis-tical analysis : the genotype frequenc+ y and allele frequency between patients with NSCL/P and control subjects were performed. Haplotype relative risk (HRR) , family based association test (FBAT) , and transmission disequilibrium test (TDT) in nuclear family were performed. Results: There were no poly-morphism in FGFR1 gene p. E467K, p. M369I and p. $393S site, the corresponding base was all G. The polymorphisms of rs13317 and rs1448037 were detected and their genotype frequency and allele frequen-cy showed no significant difference between 75 patients with NSCL/P and 75 normal children. TDT, HRR and FBAT were also no significant differences. The genotype frequency of gene FGF18 rs4043716 showed significant difference, but allele frequency were no significant difference. TDT, HRR and FBAT were also no significant difference. Conclusion: Our studies suggest an association between gene FGF18 rs4043716 and the NS CLP in Chinese population, and no association among gene FGFR1 rs13317, p. FA67K, p. M3691, p. S393S and gene FGF10 rs1448037.
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Objective To investigate the distribution and changes of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in normal heart or heart after myocardial infarction (MI),and to explore beneficial effects of bFGF on heart after MI. Methods Eleven adult minipigs were randomly divided into 2 groups,control (n=5) and MI group (n=6). MI was induced by ligating the left anterior descending coronary artery. Sham-operation was carried out on the animals of control. bFGF-like-immunoreactivity (bFGF-ir) and FGFR-1-like-immunoreactivity (FGFR-1-ir) in heart tissues were detected by immuno-histochemistry and image analysts in 4 weeks after surgery. Results In controls,bFGF-ir and FGFR-1-ir in the atrium showed a considerable high level compared with 2 ventricles (P