Effect of Huoxue Jiedu Runzao Prescription on Expression of Apoptosis Inhibitor Survivin in Submandibular Gland Cells of Sjögren's Syndrome Mice / 中国实验方剂学杂志

Objective:To investigate the effect of Huoxue Jiedu Runzao prescription on the morphology， apoptosis， and function of submandibular gland in the mouse model of Sjögren's syndrome （SS） and its functioning mechanism， we analyzed the expression of the apoptosis inhibitor Survivin in the submandibular gland cells of SS mice. Method:Female BALB/c57 mice were selected as the normal group. The naive non-obese diabetic （NOD/Ltj） female mice were selected as the SS model， which were randomly assigned into the model group， Paeoniae Radix Alba total glucosides capsule （0.234 g·kg^-1） group， and low-， medium-， and high-dose （15.6， 31.2， 62.4 g·kg^-1， respectively） Huoxue Jiedu Runzao prescription groups. Each group had 15 mice. The morphological and functional changes of submandibular gland and the Survivin expression were observed and measured after 8 weeks of drug intervention. Survivin expression was determined by immunohistochemistry （IHC）， Western blot， and reverse transcription-polymerase chain reaction （RT-PCR）. Result:Compared with normal group， salivary flow and submandibular gland index in model group were significantly decreased （<italic>P</italic><0.01）， and histopathological score of submandibular gland was significantly increased （<italic>P</italic><0.01）. Western blot showed that Survivin protein expression was significantly decreased （<italic>P</italic><0.05）. IHC showed that， Survivin mRNA expression was significantly decreased （<italic>P</italic><0.01）， and RT-PCR results showed that Survivin mRNA expression was significantly decreased （<italic>P</italic><0.01）. Compared with model group， salivary flow and submandibular gland index of mice in Huoxue Jiudu Runzao prescription groups and Paeoniae Radix Alba total glucosides capsules groups were significantly increased （<italic>P</italic><0.05）， histopathological score of submandibular gland was significantly decreased （<italic>P</italic><0.05）， IHC results showed that Survivin expression was significantly increased （<italic>P</italic><0.01）. Western blot and RT-PCR results showed that Survivin protein and mRNA expression of Huoxue Jiudu Runzao prescription high-dose group and Paeoniae Radix Alba total glucosides capsule group were significantly increased （<italic>P</italic><0.05）. Conclusion:Huoxue Jiedu Runzao prescription can improve the secretion function of submandibular acinus， increase the submandibular gland index， and saliva secretion of SS mice by up-regulating survivin in submandibular gland cells of SS mice.

Construction and screening of fibroblast-like synoviocytes in human rheumatoid arthritis stably overexpressing lncRNA HOTAIR / 医学研究生学报

Objective　The HOTAIR gene is closely related to pannus formation in rheumatoid arthritis (RA). This study aimed to construct and screen fibroblast-like synoviocytes in human RA (HFLS-RA) stably overexpressing lncRNA HOTAIR, and to pave the way for further study of the role of lncRNA HOTAIR in the pathogenesis of RA.　Methods　LncRNA HOTAIR was cloned and linked to the PMT406 vector digested by BamHI-HF-HF and XhoI. The constructed plasmids were sequenced, identified and then transfected into 293T cells to pack lentivirus. The HFLS-RA cells were infected with the recombinant and empty vector lentiviruses, and purinomycin was employed to screen the lncRNA HOTAIR-overexpressed and control cell lines. The total RNA was extracted from the blank, negatively transfected and overexpressed cells by Trizol, and the cDNA obtained by reverse transcription was amplified by qPCR, followed by determination of the expression of lncRNA HOTAIR.　Results　The relative expression of lncRNA HOTAIR was significantly higher in the overexpression group than in the blank control and negative transfection groups (30.329 ± 3.860 vs 1.001 ± 0.048 and 0.892 ± 0.247, P 0.05).　Conclusion　The HFLS-RA cell line stably overexpressing lncRNA HOTAIR was successfully constructed, which has provided some experimental evidence for further investigation of the role of lncRNA HOTAIR in the pathogenesis of RA.

Increased CD34+CD38 -CD123 + cells in myelodysplastic syndrome displaying malignant features similar to those in AML.

Leukocyte interleukin-3 receptor α (CD123) is regarded as a marker of leukemia stem cells. We previously found that CD123 was also highly expressed on CD34(+)CD38(-) cells in myelodysplastic syndrome (MDS) patients, but it is unclear whether the level and the characteristics of CD34(+)CD38(-)CD123(+) cells in MDS are similar to those in acute myeloid leukemia (AML). Based on previous research by our team, we further enlarged the specimens and found that the mean proportion and the mean MFI of CD34(+)CD38(-)CD123(+) cells in low-grade MDS were lower than that in AML, and those in high-grade MDS were similar to those in AML. CD34(+)CD38(-)CD123(+) cells expressed lower granulocyte stimulating factor receptor, CD11b, and apoptosis molecule (Annexin V), meanwhile, these cells showed upregulation of transcription factors (GATA-1, GATA-2) and transferrin receptor (CD71) in MDS and AML. Furthermore, an increase in CD34(+)CD38(-)CD123(+) cells was closely related to the number of cytopenias involving hematopoietic lineages, anemia, blast count in bone marrow smear, fluorescence in situ hybridization analysis and WHO prognostic scoring system score. Thus, increases in CD34(+)CD38(-)CD123(+) cells may reflect malignant clonal cells with aberrant differentiation, overproliferation, and decreased apoptosis in MDS, which were similar to AML. CD123 may thus be a promising indicator for identifying malignant clonal cells in MDS and a candidate for targeted therapy.

Elevated TIM3+ hematopoietic stem cells in untreated myelodysplastic syndrome displayed aberrant differentiation, overproliferation and decreased apoptosis.

TIM3, as a negative regulator of anti-tumor immunity, is highly expressed on LSCs, but not on normal HSCs. TIM3 on HSCs in MDS patients has not been clarified. Here, both the percentage of TIM3 on HSCs and the MFI of TIM3+ HSCs were higher in untreated MDS than control and were closed to AML, and excessive TIM3+ HSCs was closely related to clinical parameters: WPSS score, karyotype analysis, morphologic blasts, the number of cytopenia involving hematopoietic lineages, anemia and granulocytopenia. TIM3+ HSCs expressed lower CD11b, TpoR, EpoR, G-CSFR and Annexin V, and higher CD71 and GATA2. TIM3+ HSCs displayed aberrant differentiation, overproliferation and decreased apoptosis. TIM3 might be a promising marker for identifying malignant clone cells in MDS and a candidate for targeted therapy.

Polycythemia vera transforming to myelofibrosis and then biphenotype acute leukemia.

BACKGROUND: A case with 17-year detailed illness history including evolution of polycythemia vera (PV) to myelofibrosis (MF) and then biphenotype acute leukemia (BAL) was reported. Ten years of PV followed by seven years of MF and then BAL, the patient experienced a classical "complete course" of myeloproliferative neoplasm (MPN). High WBC counts as well as low Hb and platelet counts in MF phase, long disease course, older than 50 years age, and positive JAK2 were her high risk factors of transformation from MPN to leukemia. Pancytopenia in her secondary MF phase responded well to the therapy of corticosteroids, which indicated that the immune mechanism was involved in the pathogenesis of MF. Progression of PV to MF and then BAL might be related to discontinuation of interferon-alpha because of poor tolerance.

[TET2 and DLK1 gene expression and their clinical significance in bone marrow CD(3)(+) T cells of patients with myelodysplastic syndrome].

OBJECTIVE: To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity. METHODS: CD(3)(+) T cells were sorted by magnetic activated cell-sorting system. The expressions of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR. RESULTS: The expression of TET2 mRNA in CD(3)(+) T cells was down-regulated in the MDS patients by (0.16 ± 0.15) fold compared with the controls (P < 0.05). The expression of TET2 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with serum complement C(3) (r = 0.404, P < 0.05). The expression of DLK1 mRNA in CD(3)(+) T cells was up-regulated in the MDS patients by (1.61 ± 0.88) folds compared with the controls (P < 0.05). Grouped by the chromosomes, the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [(1.45 ± 0.44) folds, P < 0.05]. The expression of DLK1 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with the proportion of bone marrow blasts (r = 0.343, P < 0.05). CONCLUSIONS: The mRNA expression of TET2 in CD(3)(+) T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased, which might decline the immune surveillance function. The findings would be useful for exploring the mechanism of immune tolerance.

[STAT5 phosphorylation in CD34(+)CD38(-)CD123(+) bone marrow cells of the patients with myelodysplastic syndrome].

OBJECTIVE: To investigate the expressions of STAT5 phosphorylation in CD34(+)CD38(-)CD123(+) bone marrow cells of the patients with myelodysplastic syndromes (MDS), and then evaluate the level of activation of STAT5 associated with cell proliferation in MDS clone cells. METHODS: The bone marrow mononuclear cells (BMMNC) were extracted from 36 MDS patients and 14 normal controls. The mean fluorescence intensities (MFI) of phosphorylated STAT5(P-STAT5) in CD34(+)CD38(-)CD123(+) and CD34(+)CD38(-)CD123(-)cells, with or without the stimulation of 10 U/ml EPO, were examined by flow cytometry (FCM). RESULTS: Without stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 113.71 ± 67.22/173.05 ± 102.78, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (58.84 ± 27.51/68.99 ± 50.42, P < 0.01, P < 0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (63.06 ± 21.06, P < 0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; With the EPO stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 144.04 ± 58.11/239.45 ± 152.05, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (68.41 ± 25, 10/64.21 ± 23.43, P < 0.01) and the normal controls CD34(+)CD38(-)CD123(-) cells (75.21 ± 27.02, P < 0.01), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; The P-STAT5 MFI in the CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients with or without EPO stimulation were 21.80/28.86, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (7.42/5.50, P < 0.01, P < 0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (6.39, P < 0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal controls CD34(+)CD38(-)CD123(-) cells; There was no significant difference of P-STAT5 MFI with or without EPO stimulation and the increased P-STAT5 MFI between the CD34(+)CD38(-)CD123(+) cells of low and high risk MDS. CONCLUSION: STAT5 associated with cell proliferation was activated in CD34(+)CD38(-)CD123(+) bone marrow cells in MDS, which had more significant reactions to EPO than CD34(+)CD38(-)CD123(-) cells, indicating that CD34(+)CD38(-)CD123(+) bone marrow cells might be the real malignant MDS clone cells in MDS.

Research progress on the red cell diseases in China.

In recent years, there have been lots of progresses in the studies on red cell diseases in China, especially bone marrow failure diseases including immuno-related pancytopenia, aplastic anemia, myelodysplastic syndrome, and paroxymal nocturnal hemoglobinuria. Numerous laboratory experiments as well as clinical researches have been carried out by Chinese hematologists, which brought about much clearer pathogenesis, more rational diagnosis methods and more effective therapies for red cell diseases.

Expression of DLK1 Gene in the Bone Marrow Cells of Patients with Myelodysplastic Syndromes and Its Clinical Significance.

OBJECTIVE: This study aims to investigate the expression of delta-like 1 (DLK1) gene in the bone marrow cells of patients with myelodysplastic syndromes (MDS) and to explore its molecular characteristics for the early diagnosis of MDS. METHODS: The expression of DLK1 mRNA in the bone marrow cells of cases with MDS, acute myeloid leukemia (AML), and normal control groups were measured by real-time polymerase chain reaction and were analyzed for clinical significance. RESULTS: Significantly higher expression of DLK1 mRNA was observed in the bone marrow cells of MDS patients (0.7342±0.3652) compared with the normal control group (0.4801±0.1759) (P<0.05). The expression of DLK1 mRNA had a positive correlation with the proportion of bone marrow blasts (r=0.467, P<0.05). Moreover, DLK1 mRNA expression was significantly increased as MDS progressed (P<0.05). Patients with abnormal karyotypes exhibited significantly higher expression of DLK1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Subsequently, patients with highly expressed DLK1 (≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower DLK1 expression (<0.8),(0.1517±0.3109), (P<0.05). CONCLUSIONS: The DLK1 gene was highly expressed in MDS patients, and was increased as MDS progressed. The expression of DLK1 mRNA was positively correlated with the proportion of the bone marrow blasts. A high expression of DLK1 gene suggested a higher malignant clone burden of MDS.

[Differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes].

OBJECTIVE: To detect the abnormalities of differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes (MDS). METHODS: Forty-five newly diagnosed MDS cases from July 2008 to March 2010 in our hospital and 30 normal controls were enrolled. There were 17 low-risk and 28 high-risk patients. The CD34+CD38+ and CD34+CD38- bone marrow cells and the expressions of stem cell factor receptor (SCF-R), erythropoietin receptor (EpoR), granulocyte colony-stimulating factor receptors (G-CSFR) and thrombopoietin receptor (TpoR) on those cells were measured by flow cytometry. RESULTS: The mean percentage of CD34+ in karyocyte of MDS cases in high-risk patients [0.53% (0.10%-1.68%)] was significantly higher than that of control group [0.13% (0.08%-0.32%), P<0.01]. The mean percentages of CD34+CD38+ cells were significantly lower in low and high-risk groups (86.3%±8.5% and 82.6%±11.1%) than those in control group (92.3%±3.4%). And the percentage of CD34+CD38- cells was significantly higher in either low-risk or high-risk group (13.7%±8.5% and 17.4%±11.0%) than that in control group (7.7%±3.4%, both P<0.05). In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+ cells than that in CD34+CD38- cells (18.7%±18.3% vs 63.6%±20.0%, P<0.01). The expressions of SCF-R, G-CSFR and TpoR were not significantly different between two cell populations. The expressions of EpoR on CD34+CD38+ cells of low and high-risk MDS groups [9.0% (1.4%-12.7%), 5.2% (1.1%-14.1%)] were significantly lower than those of control group [9.6% (5.1%-30.1%), both P<0.05]. The expressions of G-CSFR on CD34+CD38+ cells of low and high-risk MDS groups (29.8%±19.1%, 28.7%±21.1%) were significantly lower than those of control group (44.4%±23.4%, both P<0.05). The quantities of EpoR on CD34+CD38- cells of low and high-risk MDS groups (42.2%±21.9%, 25.7%±15.6%) were significantly lower than those of control group (63.6%±20.0%, both P<0.01). The expressions of TpoR on CD34+CD38- cells of low and high-risk MDS groups (5.4%±4.7%, 4.1%±4.0%) were significantly lower than those of control group (10.1%±8.3%, both P<0.05). The incidence of cytopenia with low expression rates of hemopoietic cytokine receptors on CD34+ cells was higher than that of MDS with high expression rates. CONCLUSION: The abnormalities of differentiation and membrane hemopoietic cytokine receptors expression of CD34+ bone marrow cells in MDS are associated with MDS cytopenia and may be useful for the diagnosis of MDS.

Expression of CD123 and CD114 on the bone marrow cells of patients with myelodysplastic syndrome.

BACKGROUND: Recent studies have shown that interleukin-3 receptor alpha (CD123) is highly expressed on leukemia stem cells of patients with acute myeloid leukemia, and is correlated with tumor load and poor prognosis. The expression of CD123 may also be high in patients with myelodysplastic syndrome (MDS). In this study, the expression and clinical significance of CD123 and granulocyte colony stimulating factor (G-CSF) receptor (CD114) on the bone marrow cells of patients with MDS were investigated to explore the molecular marker of the malignant clone of MDS. METHODS: Forty-two patients with MDS, who were diagnosed in the Hematological Department of General Hospital of Tianjin Medical University from 2008 to 2009, and twelve normal controls were enrolled in this study. Fluorescence activiated cell sorter (FACS) was used to measure the expression of CD123 on CD34(+)CD38(-) cells and CD114 on CD34(+) cells of the bone marrow of these patients and controls and the clinical significance was analyzed. The expression of CD114 on CD123(+)CD34(+)CD38(-) cells was further measured to explore the molecular marker of the malignant clone in MDS. RESULTS: MDS patients displayed significantly higher proportion of CD34(+)CD38(-)/CD34(+) ((14.03 +/- 5.27)%) than normal controls ((7.70 +/- 4.36)%, P < 0.05). The expression rate of CD123(+)CD34(+)CD38(-)/CD34(+)CD38(-) was significantly higher in MDS patients ((48.39 +/- 28.15)%) than that in normal controls ((8.75 +/- 11.71)%, P < 0.01). The expression level of CD123 was significantly correlated with the proportion of bone marrow blasts (r = 0.457, P < 0.05). The expression rate of CD114(+)CD34(+)/CD34(+) was lower in MDS patients ((33.05 +/- 21.71)%) than that in normal controls ((38.99 +/- 19.07)%) but was not statistically significant (P > 0.05). The expression of CD114 on CD123(+)CD34(+)CD38(-) cells ((34.82 +/- 29.58)%) was significantly lower than that on CD123(-)CD34(+)CD38(-) cells ((53.48 +/- 27.41)%) of MDS patients (P < 0.05). CONCLUSIONS: MDS patients displayed higher proportion of CD34(+)CD38(-)/CD34(+) than normal controls. CD123 was highly expressed in the bone marrow of the patients with MDS, significantly correlated with the proportion of bone marrow blasts, and thus might be the marker of MDS malignant clone. CD123(+)CD34(+)CD38(-) cells exhibited lower expression of G-CSF receptors, which might partly explain why MDS clone responds worse to G-CSF in vitro and in vivo.

[Expression of differentiation antigens on bone marrow myeloid cells of the patients with myelodysplastic syndromes and its clinical significances].

OBJECTIVE: To detect the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients. METHODS: Quantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality and the prognosis of MDS cases were analyzed. RESULTS: The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The "right hook", "sickle" and "retroflex 7" shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+ (0.39 +/- 0.34) and CD16-/CD16+ (1.33 +/- 0.77) were significantly higher in high-risk MDS group than those of control group (0.07 +/- 0.05 and 0.39 +/- 0.31 respectively) (P < 0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ 3.88% +/- 3.07%, CD11b-HLA-DR- 16.23% +/- 15.59%, CD16-HLA-DR- 41.12% +/- 24.53%, CD11b+CD16- 33.53% +/- 17.26% and CD13+CD16- 44.51% +/- 21.99% granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P < 0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases per case correlated directly with their IPSS (international prognostic scoring system) (r = 0.690, P = 0.000) and WPSS (WHO adapted prognostic scoring system) (r = 0.651, P = 0.000) scores. CONCLUSION: There is an abnormal expression of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity is correlated with the prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.

[Abnormalities of CD34+ cells differentiation and bone marrow cell cycle in myelodysplastic syndrome].

OBJECTIVES: To detect the abnormalities of CD(34)(+) cells differentiation and bone marrow cell cycle in myelodysplastic syndrome (MDS). METHODS: Fifty newly diagnosed MDS (17 in low risk and 33 in high risk), 8 acute myeloid leukemia preceded by MDS (MDS-AML) and 25 normal controls were enrolled into this study. Their CD(34)(+)CD(38)(+), CD(34)(+)CD(38)(-) bone marrow cells and bone marrow cell cycle were measured with flow cytometry. RESULTS: The mean percentages of CD(34)(+) cells in bone marrow karyocyte of high risk [(2.29 ± 2.17)%] and MDS-AML groups [(18.69 ± 17.47)%] were significantly higher than that of control group [(0.36 ± 0.49)%, P < 0.05]. The mean percentages of CD(34)(+)CD(38)(+) cells were significantly lower in low risk, high risk and MDS-AML groups [(86.09 ± 7.79)%, (81.68 ± 11.82)% and (82.88 ± 2.60)%, respectively] than that in control group [(92.21 ± 3.85)%, P < 0.05], thus the percentages of CD(34)(+)CD(38)(-) cells were significantly higher in either MDS (low risk and high risk) or MDS-AML groups [(13.91 ± 7.79)%, (18.32 ± 11.82)% or (17.13 ± 2.60)%, respectively] than that in control group [(7.79 ± 3.85)%, P < 0.05]. The percentages of CD(34)(+)CD(38)(-) cells of MDS cases correlated directly with their International Prognostic Scoring System (IPSS) (r = 0.493, P = 0.001) and WHO Adapted Prognostic Scoring System (WPSS) (r = 0.586, P = 0.000) scores. The percentages of bone marrow mononuclear cells (BMMNCs) in G(0)/G(1) phase of in low risk, high risk and MDS-AML groups [(94.52 ± 4.32)%, (96.07 ± 3.88)% and (94.65 ± 4.55)%, respectively] were significantly higher than that in control group [(88.94 ± 7.30)%, P < 0.01], thus the percentages of BMMNCs in S and G(2)/M phase were significantly lower in either MDS (low risk and high risk) or MDS-AML groups than that in control group (P < 0.05). MDS patients with low percentages of CD(34)(+)CD(38)(-) cells presented higher therapeutic efficacy than those with high percentages of CD(34)(+)CD(38)(-) cells, while without significant differences (P > 0.05). CONCLUSIONS: There are abnormalities of differentiation of CD(34)(+) bone marrow cells and high proportion of G(0)/G(1) cells which indicates a G(1) phase arrest in MDS that might be involved in the pathogenesis of MDS. So the examination of CD(34)(+) bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effects.