[The Effect and Safety of Flumatinib in Patients with Chronic Myelogenous Leukemia Failed First-and Second-line Treatment].

OBJECTIVE: To analyze the efficacy and safety of flumatinib, a second-generation tyrosine kinase inhibitor (TKI) independently developed in China, in patients with chronic myelogenous leukemia in chronic phase (CML-CP) who falied first-line and second-line treatment. METHODS: The clinical data of 30 CML-CP patients treated with flumatinib in Lianyungang First People's Hospital from January 2020 to September 2022 were collected retrospectively. Among them, 15 patients who received imatinib first-line treatment but failed treatment were included in the second-line group, and the other 15 patients who failed second-line treatment with nilotinib or dasatinib were included in the third-line group. The hematological and molecular responses of the patients in the two groups at 3, 6 and 12 months of treatment, and the event-free survival (EFS) and adverse reactions of patients at the end of follow-up were statistical analyzed. RESULTS: At 3, 6, and 12 months of treatment, 10, 11, and 12 patients in the second line group achieved major molecular response (MMR), which was higher than that of 3, 4, and 5 patients in the third line group (P =0.010, P =0.011, P =0.010). At 3 months of treatment, 12 and 13 patients achieved complete hematological response (CHR) and early molecular response (EMR) in the second-line group, which was higher than that of 9 and 13 patients in the third-line group, but the difference between the two groups was not statistically significant (P =0.232, P =1.000); At 6 and 12 months of treatment, 6 and 7 patients in the second-line group achieved MR4.5, which were higher than of 3 and 2 cases in the third-line group, but the difference was not statistically significant (P =0.427, P =0.713). The hematological adverse reactions of patients in the second-line group during treatment the period were mainly grade 1-2 thrombocytopenia and anemia, and no grade 3-4 of adverse reactions occurred. In the third-line group, there were 2 cases of grade 1-2 thrombocytopenia, grade 1-2 anemia and white blood cell 3 cases were reduced each, 1 case of grade 3-4 anemia, 2 cases of grade 3-4 neutropenia. The non-hematological adverse reactions in the second-line group were rash (2 cases), headache (1 case), diarrhea (1 case), fatigue (1 case), limb pain (1 case). There were 1 cases of diarrhea, 1 cases of nausea, and 1 cases of edema in the third-line group. There was no statistical significance in hematological and non-hematological adverse reactions between the two groups of patients (P >0.05). At the end of follow-up, the EFS rate of patients in the second-line group was higher than that in the third-line group (100% vs 93.3%), but the difference was not statistically significant (P =0.317). CONCLUSION: The second-generation TKI flumatinib independently developed in China, has good curative effect and safety for CML-CP patients who failed first-line and second-line treatment.

[Clinical Efficacy and Safety of Ixazomib-Containing Regimens in the Treatment of Patients with Multiple Myeloma].

OBJECTIVE: To investigate the clinical efficacy and safety of ixazomib-containing regimens in the treatment of patients with multiple myeloma (MM). METHODS: A retrospective analysis was performed on the clinical efficacy and adverse reactions of 32 MM patients treated with a combined regimen containing ixazomib in the Hematology Department of the First People's Hospital of Lianyungang from January 2020 to February 2022. Among the 32 patients, 15 patients were relapsed and refractory multiple myeloma (R/RMM) (R/RMM group), 17 patients who responded to bortezomib induction therapy but converted to ixazomib-containing regimen due to adverse events (AE) or other reasons (conversion treatment group). The treatment included IPD regimen (ixazomib+pomalidomide+dexamethasone), IRD regimen (ixazomib+lenalidomide+dexamethasone), ICD regimen (ixazomib+cyclophosphamide+dexamethasone), ID regimen (ixazomib+dexamethasone). RESULTS: Of 15 R/RMM patients, overall response rate (ORR) was 53.3%(8/15), among them, 1 achieved complete response (CR), 2 achieved very good partial response (VGPR) and 5 achieved partial response (PR). The ORR of the IPD, IRD, ICD and ID regimen group were 100%（3/3）, 42.9%（3/7）, 33.3%（1/3）, 50%（1/2）， respectively， there was no statistically significant difference in ORR between four groups (χ 2=3.375, P =0.452). The ORR of patients was 50% after first-line therapy, 42.9% after second line therapy, 60% after third line therapy or more, with no statistically significant difference among them (χ2=2.164, P =0.730). In conversion treatment group, ORR was 88.2%(15/17), among them, 6 patients achieved CR, 5 patients achieved VGPR and 4 patients achieved PR. There was no statistically significant difference in ORR between the IPD（100%， 3/3）, IRD（100%， 6/6）, ICD（100%， 3/3） and ID（60%， 3/5） regimen groups (χ2=3.737，P =0.184). The median progression-free survival (PFS) time of R/RMM patients was 9 months (95% CI : 6.6-11.4 months), the median overall survival (OS) time was 18 months (95% CI : 11.8-24.4 months). The median PFS time of conversion treatment group was 15 months (95% CI : 7.3-22.7 months), the median OS time not reached. A total of 10 patients suffered grade 3- 4 adverse event (AE). The common hematological toxicities were leukocytopenia, anemia, thrombocytopenia. The common non-hematological toxicities were gastrointestinal symptoms (diarrhea, nausea and vomit), peripheral neuropathy, fatigue and infections. Grade 1-2 peripheral neurotoxicity occurred in 7 patients. CONCLUSION: The ixazomib-based chemotherapy regimens are safe and effective in R/RMM therapy, particularly for conversion patients who are effective for bortezomib therapy. The AE was manageable and safe.

[Efficacy and Safety of Decitabine Combined with Modified EIAG Regimen in the Treatment of Patients with Relapsed/Refractory Acute Myeloid Leukemia and High-risk Myelodysplastic Syndrome].

OBJECTIVE: To investigate the efficacy, prognosis and safety of decitabine combined with modified EIAG regimen in the treatment of patients with relapsed/refractory acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). METHODS: The clinical data of 44 patients with relapsed/refractory AML and high-risk MDS admitted to our hospital from January 2017 to December 2020 were analyzed retrospectively. The patients were equally divided into D-EIAG group (decitabine combined with EIAG regimen) and D-CAG group (decitabine combined with CAG regimen) according to clinical treatment regimen. The complete response (CR), CR with incomplete hematologic recover (CRi), morphologic leukemia-free state (MLFS), partial response (PR), overall response rate (ORR), modified composite complete response (mCRc), overall survival (OS) time, 1-year OS rate, myelosuppression and adverse reactions between the two groups were compared. RESULTS: In D-EIAG group, 16 patients (72.7%) achieved mCRc (CR+CRi+MLFS), 3 patients (13.6%) achieved PR, and ORR (mCRc+PR) was 86.4%. In D-CAG group, 9 patients (40.9%) achieved mCRc, 6 patients (27.3%) achieved PR, and ORR was 68.2%. Difference was observed in mCRc rate between the two groups (P=0.035), but not in ORR (P>0.05). The median OS time of D-EIAG group and D-CAG group was 20 (2-38) months and 16 (3-32) months, and 1-year OS rate was 72.7% and 59.1%, respectively. There was no significant difference in 1-year OS rate between the two groups (P>0.05). After induction chemotherapy, the median time for absolute neutrophil count recovery to 0.5×109/L in D-EIAG group and D-CAG group was 14 (10-27) d and 12 (10-26) d, for platelet count recovery to 20×109/L was 15 (11-28) d and 14 (11-24)d, the median red blood cell suspension transfusion volume was 8 (6-12) U and 6 (6-12) U, and the median apheresis platelet transfusion volume was 4 (2-8) U and 3 (2-6) U, respectively. There were no statistically significant differences in comparison of the above indicators between the two groups (P>0.05). The hematological adverse reactions of patients were mainly myelosuppression. Grade III-IV hematological adverse events occurred in both groups (100%), with no increase in the incidence of non-hematological toxicities such as gastrointestinal reactions or liver function damage. CONCLUSION: Decitabine combined with EIAG regimen in the treatment of relapsed/refractory AML and high-risk MDS can improve remission rate, provide an opportunity for subsequent therapies, and have no increase in adverse reactions compared with D-CAG regimen.

[Analysis of Related Factors Affecting the Nosopoiesis of Childhood Acute Leukemia].

OBJECTIVE: To analyze the related factors affecting the nosopoiesis of childhood acute leukemia from the perspective of indoor environmental exposure, behavior and lifestyle. METHODS: The clinical data of 64 children with acute leukemia were retrospectively analyzed, and 50 healthy children were selected as the control group during the same period. The basic data of children, indoor environment, behavior and lifestyle of parents in 2 groups were recorded. Univariate and multivariate logistic regression analysis were used to analyze the related factors affecting the incidence of childhood acute leukemia, and the OR (95%CI) value was calculated. RESULTS: The unvariate analysis showed that the daily wine-drinking rate of father and pesticide use rate in acute leukemia group were significantly higher than those in control group (P<0.05). Multivariate Logistic regression analysis showed that indoor ventilation during summer sleep of children (OR=0.35, 95%CI: 0.14-0.88) and contact with other children before 3 years old (OR=0.34, 95%CI: 0.18-0.65) were protective factors for provention of childhood acute leukemia (P<0.05). Mothers had a history of exposure to chemical substances (OR=3.68, 95%CI: 1.64-8.27), and children had a history of exposure to chemical substances (OR=3.84, 95%CI: 1.64-9.01), family had internal decoration history after child birth (OR = 1.38, 95%CI: 1.05-1.81) and family uses of pesticides (OR=2.17, 95%CI: 1.08-4.36), all these factors were independent risk factors for acute leukemia (P<0.05). CONCLUSION: Indoor environmental exposure, behavior and lifestyle of children and parents may be closely related with the nosopoiesis of childhood acute leukemia.

Regenerating infected bone defects with osteocompatible microspheres possessing antibacterial activity.

Treatment of infected bone defects still remains a formidable clinical challenge, and the design of bone implants with both anti-bacterial activity and -osteogenesis effects is nowadays regarded as a powerful strategy for infection control and bone healing. In the present study, bioresorbable porous-structured microspheres were fabricated from an amphiphilic block copolymer composed of poly(l-lactide) and poly(ethyl glycol) blocks. After being surface coated with mussel-inspired polydopamine, the microspheres were loaded with nanosilver via the reduction of silver nitrate and apatite via biomineralization in sequence. At optimized loading amounts, the nanosilver-loaded microspheres showed no unfavorable effects on the proliferation and differentiation of bone marrow mesenchymal stem cells despite preserving strong antibacterial activity in in vitro evaluations. For the critical-sized defects (φ = 8 mm) in the rat cranium that was pre-infected with Staphylococcus aureus, the filling of the dual-purpose microspheres demonstrated an effective way to kill bacteria in vivo, and in the meantime, it promoted new bone formation efficiently alongside the degradation of microspheres. Thus, the results suggested that bioresorbable microspheres with both osteoconductive and antibacterial activities were a good choice for treating infected bone defects.

Computer-assisted, minimally invasive transforaminal lumbar interbody fusion: One surgeon's learning curve A STROBE-compliant article.

Minimally invasive (MI) transforaminal lumbar interbody fusion (TLIF) is a challenging technique with a long learning curve. We combined computer-assisted navigation and MI TLIF (CAMISS TLIF) to treat lumbar degenerative disease. This study aimed to evaluate the learning curve associated with computer-assisted navigation MI spine surgery (CAMISS) and TLIF for the surgical treatment of lumbar degenerative disease. Seventy four consecutive patients with lumbar degenerative disease underwent CAMISS TLIF between March 2011 and May 2015; all surgeries were performed by a single surgeon. According to the plateau of the asymptote, the initial 25 patients constituted the early group and the remaining patients comprised the latter group. The clinical evaluation data included operative times, anesthesia times, intraoperative blood losses, days until ambulation, postoperative hospital stays, visual analog scale (VAS) leg and back pain scores, Oswestry disability index (ODI) values, Macnab outcome scale scores, complications, radiological outcomes, and rates of conversion to open surgery. The complexity of the cases increased over the series, but the complication rate decreased (12.00%-6.12%). There were significant differences between the early and late groups with respect to the average surgical times and durations of anesthesia, but no differences in intraoperative blood losses, days until ambulation, postoperative hospital stays, complication rate, VAS, ODI, Macnab outcome scale scores, or solid fusion rates. There was no need for conversion to open procedures in either group. Our study showed that a plateau asymptote for CAMISS TLIF was reached after 25 operations. The later patients experienced shorter operative times and anesthesia durations.

Three-dimensional spheroid culture promotes the stemness maintenance of cranial stem cells by activating PI3K/AKT and suppressing NF-κB pathways.

Multipotent stem cells are one of the most powerful tools available for the bone regeneration. However, owing to various limitations, including a lack of tissue-specific stem cell identification, reconstruction of large cranial bone defects remains challenging. In the current study, we isolated a population of Sca-1+CD105+CD140a+ stem cells from adult mouse calvarium and cultured them as three-dimensional spheroids. Although these cells shared similar surface antigens when grown in either monolayers or spheroids, the cranial stem cells grown in spheroids possessed enhanced multipotency and proliferation capacity. In addition, the cranial stem cells in spheroids were found to express high levels of the self-renewal transcription factors Nanog, Oct-4, and Sox-2. Mechanistically, we found that three-dimensional spheroid culture suppressed NF-κB pathways, but activated the PI3K/AKT pathway in cranial stem cells. More importantly, activation of NF-κB pathways or specific inhibition of the PI3K/AKT pathway partially impaired spheroid formation and suppressed expression of self-renewal transcription factors. In summary, these findings reveal a novel effect of spheroid culture in promoting the maintenance of cranial stem cell stemness and indicate that NF-κB and PI3K/AKT pathways might be involved in the stemness maintenance.

[IDH1 Gene Mutation and Its Clinical Significance in patients with Acute Myeloid Leukemia].

OBJECTIVE: To evaluate the incidence rate of IDH1 in acute myeloid leukemia and analyze its effect on clinical characteristics and prognosis. METHODS: Mononuclear cells in bone marrow samples were collected from 192 adult patients with newly diagnosed AML. Polymerase chain reaction (PCR) and direct sequencing were used to amplify exon 4 of IDH1 gene, the gene sequencing was used to analyze the gene mutations, at same time, the detection of NPM1, FLT3-TKD, FLT3-ITD, C-KIT, CEPBA, TET2 and JAK2V617F and MLL mutations were carried out, the follow-up was used to determine its therapeutic efficacy and outcomes of patients. The clinical and laboratory data of these cases were collected, and their clinical characteristics and prognosis were then analyzed. RESULTS: Among the 192 AML patients, 13 cases were detected with IDH1 gene mutation, the mutation rate was 6.77% [95% CI (5.70%-13.38%)]. The sequencing chart of IDH1 gene showed double peaks, the mutations were heterozygous, out of them c.G395A (p.R132H) was found in 8 cases, c.C394T was found in 4 cases (p.R132C), c.C394A (p.R132S) was found in 1 cases, R132H and R132C are common, 13 cases showed missense mutation. The median age in mutation group was 52 years old, the median age in unnutration group was 40 years, there was significant difference between them (P = 0.010). Mutation rate of IDH1 gene in M1 and M2 was significantly higher than that in other FAB subtypes. There were no significant difference in sex, newly diagnosed peripheral white blood cell count, hemoglobin, platelet count, peripheral blood and bone marrow original cell proportion of primitive cells between them. Mutation of IDH1 gene had certain correlation with NPM1 gene mutation, but no correlation with FLT3-TKD, FLT3-ITD, C-KIT, TET2 and JAK2V617F and MLL natations was found. In addition, the IDH1 mutation easily occurred in patients with normal karyotype or in patients with middle prognostic risk karyotype, IDH1 mutation occurred in 11 cases with normal karyotype, the mutation rate was 10.28%, IDH1 mutation were observed in 2 cases with abnormal karyotype, the mutation rate was 3.50%, there was significant difference. In AML patients with middle prognostic risk karyotype. The complete remission (CR) and the 3 year survival (OS) rate of IDH1 mut patients were less than that in IDH1 wt, there was significant difference (P < 0.05). CONCLUSIONS: The IDH1 mutation more easily occurr in older AML patients and mutations effect of IDH1 on clinical characteristics may represent a molecular marker for poor prognosis in AML.

Reversal of anterior cervical discectomy and fusion with cervical artificial disc replacement: regain motion after 9 years fusion.

STUDY DESIGN: Case report and review of the literature. OBJECTIVE: To present a conversion of an anterior cervical discectomy and fusion (ACDF) with a cervical artificial disc replacement in a 39-year-old woman and to review the relevant literature. SUMMARY OF BACKGROUND DATA: Return of functional spinal unit motion 9 years after attempted fusion is extremely rare. METHODS: The patient underwent an attempted anterior cervical discectomy and fusion 9 years earlier for bilateral hand numbness and leg weakness. Most of her neurological deficits had resolved after the index operation, but returned 2 months before the second operation and were unresponsive to nonoperative treatment. Computed tomography (CT) myelography showed recurrence of cervical disc herniation at the cephalad adjacent segment, which compressed the spinal cord. There was still some osteophyte at the C5/6 level that was also causing compression to the spinal cord. A solid fusion was suspected at this level. Surgery was performed to take down the grafted region and replace both levels with artificial disks. RESULTS: The range of motion (ROM) of the revised level at the 6-month follow-up was well preserved, there was no sign of instability at either operated level. The 6-month follow-up CT scan shows that, there was no obvious compression in the spinal canal. The remobilized facet joints of C5/6 segment have not demonstrated further degeneration. The patient's neck symptom and neurological function were significantly recovered. CONCLUSIONS: This case demonstrates application of a cervical artificial disc replacement to restore motion at a level that was previously grafted and fused. In select cases, cervical artificial disc replacement may represent a reasonable alternative to a repeated attempt at fusion. It is imperative that preoperatively lack of fusion of the facet joints be demonstrated on reconstructed CT scanning.

[Longitudinal spinous splitting laminoplasty with coral bone for cervical stenosis].

OBJECTIVE: To explore the outcomes of longitudinal spinous splitting laminoplasty with coral bone (abbreviated as SLAC) for cervical stenosis. METHODS: A total of 142 patients underwent conventional SLAC while 147 other patients modified SLAC. Assessments were made at pre-operation, post-operation and 3-month follow-up to examine the effects of two surgical approaches on the recovery rate of JOA (Japanese Orthopedic Association) score. The change of cervical alignment, change of cervical motions, axial syndrome, operative duration and intra-operative blood loss were recorded and analyzed with SPSS 13.0. RESULTS: No significant difference existed between two groups in the recovery rate of JOA score and intra-operative blood loss. The smaller change of cervical alignment and change of cervical motions were found in the modified SLAC group. The modified SLAC group had fewer patients with axial syndrome during the follow-up period. The operative duration was shorter in the modified SLAC group. CONCLUSION: Preventing muscle injuries in cervical laminoplasty can reduce the incidence of complications. The modified SLAC approach may protect cervical posterior extensor musculature, maintain the cervical lordotic alignment and reduce the incidence of post-operative axial syndrome.

[One-stage posterior approach spinal reconstruction for severe lumbar burst fracture].

OBJECTIVE: To evaluate the clinical therapeutic outcomes for severe burst fracture of lumbar vertebrae via a posterior approach for one-stage spinal "three-column" reconstruction. METHODS: An retrospective analysis of data was performed for 21 patients with severe burst fracture of lumbar vertebrae undergoing a posterior approach for one-stage spinal "three-column" reconstruction from 2005 to 2010. It was compared with previous 15 cases undergoing a staged anteroposterior approach. The operative duration, volume of blood loss, pre- and post-operative imaging measurements of kyphosis and vertebral height and nerve function recovery were evaluated. RESULTS: The values of operative duration and volume of blood loss in the one-stage posterior approach group were significantly less than those of the two-stage anteroposterior approach group [(263 ± 72) min vs (439 ± 75) min, t = -5.303, P < 0.01; (1143 ± 296) ml vs (1471 ± 399) ml, t = -2.169, P = 0.042)]. Statistical significance existed in postoperative kyphosis between two groups [(0.5 ± 2.0)° vs (3.9 ± 2.6)°, t = -3.336, P = 0.003]. Vertebral height had no statistical significance pre- and post-operatively between two groups while restoration of vertebral height did [(0.47 ± 0.19) mm vs (0.26 ± 0.15) mm, t = 2.669, P = 0.015]. CONCLUSION: Posterior approach for one-stage vertebral resection, mesh implantation, pedicle screws and rod internal fixation for reconstructing spinal "three-column" structures offers excellent feasibility and safety. And it may avoid complications associated with an anteroposterior approach for two-stage procedures. The median length of hospital stay is also reduced.

[Short-term clinical outcome of modified expansive open-door cervical laminoplasty preserving posterior extensor musculature inserted into C2 and C7 spinous process].

OBJECTIVE: To assess the effect of modified cervical expansive open-door laminoplasty preserving the posterior extensor musculature inserted into the C2 and C7 spinous process upon maintaining the cervical lordotic alignment and axial syndrome and to determine whether preserving the posterior extensor musculature inserted into C2 and C7 spinous process can reduce the complications. METHODS: Twenty-eight patients undergoing modified cervical expansive open-door laminoplasty preserving the posterior extensor musculature inserted into the C2 and C7 spinous process and 21 patients undergoing conventional C3-C7 cervical expansive open-door laminoplasty were investigated in pre-operative, post-operative and 3-month follow-up. The investigators assessed the effects of two different cervical laminoplasty types in the recovery rate of JOA score, the changes of Cobb angle and Ishihara's index, axial syndrome, operating duration and intra-operative blood loss, analyzed the results in SPSS and tried to find the difference in two operative types. RESULTS: There were the same results in the recovery rate of JOA score and intra-operative blood loss in modified expansive open-door cervical laminoplasty group and the conventional C3-C7 cervical expansive open-door laminoplasty group. The smaller changes of Cobb angle and Ishihara's index in the follow-up of modified laminoplasty group were found. The modified laminoplasty group had fewer patients suffering the axial syndrome in follow-up. The operating duration was shorter in the modified laminoplasty group. CONCLUSION: Preventing muscle injuries in cervical laminoplasty can reduce the incidence of complications. The modified expansive open-door cervical laminoplasty preserving the posterior extensor musculature inserted into C2 and C7 spinous process can protect cervical posterior extensor musculature. This is helpful to maintain the cervical lordotic alignment and reduce the incidence of post-operative axial syndrome.

[Effects and mechanism of CD4+ CD25+ regulatory T cells in mouse experimental colitis treated by CLYSTER No. 1].

OBJECTIVE: To explore the effects and mechanism of CD4+ CD25+ regulatory T cells (Tregs) in mouse experimental colitis treated by CLYSTER No. 1. METHOD: The mouse model of experimental colitis was established by dinitrochlorobenzene (DNCB)-acetic acid (AA) in mice DNCB and AA. Adult KM mouse were randomly divided into four groups: normal control group, experimental colitis model group, SASP and Chinese medicine therapeutic groups. Proportion of CD4 CD25+ Tregs in peripheral blood (PB) and mesenteric lymph node (MLN) was estimated by flow cytometry at the end of one or two week after treating with SASP and CLYSTER No. 1. RESULT: The model of experimental colitis in mouse was successfully established. Compared with normal control group, the proportion of CD4 CD25 Tregs was markedly decreased in PB and MLN of model control group of experimental colitis. But it was significantly increased in therapeutic groups of SASP and CLYSTER No. 1, and their CD4+ CD25+ Tregs in PB and MLN were much more than the model control group at the end of one or two weeks after treating with SASP and CLYSTER No. 1. CONCLUSION: CD4+ CD25+ Tregs with strong immune suppression could play a central role in the initiation and development of mouse experiment colitis, and the CLYSTER No. 1 might exert its therapeutic effects on UC by the regulation of number and function of CD4+ CD25+ Tregs.

[Effect of RNA interference for Lon gene silencing on growth and apoptosis of human breast cancer MCF7 cells].

OBJECTIVE: To study the role of Lon gene in tumor cell proliferation, apoptosis and cell stress response. METHODS: Small interfering RNAs (smRNAs) for Lon gene were designed using Ambion software and synthesized. The recombinant plasmid pSilencer U6 2.1/Lon was constructed with the smRNAs and pSilencer U6 2.1, followed by transfection into MCF7 cells via Lipofectamine(TM) 2000. The positive cLones were detected by RT-PCR 24 h after cell transfection. The transfected MCF7 cells were then subjected to cisplatin treatment, ultraviolet (UV) exposure and heat stress, respectively, after which the cells growth was tested with MTT assay and the measurements were plotted against time or concentration depending on the treatment administered. Apoptosis of MCF7 cells following the treatments was measured with flow cytometry. RESULTS: The mRNA of Lon gene was downregulated in cells transfected with the recombinant plasmid pSilencer U6 2.1-Lon, and RT-PCR fail to detect the specific band of Lon as could be detected in untransfected and mock-transfected MCF7 cells. MTT assay showed that pSilencer U6 2.1-Lon transfection resulted in reduced cell proliferation capacity. Stress response test revealed that MCF7 cells with Lon gene down-regulation enhanced cell sensitivity for UV and cisplatin, which was not observed for non-transfected or mock transfection group. The same changes were also observed for heat stress exposure at 41 degrees Celsius;, but not at 43 degrees Celsius; or 45 degrees Celsius;. Increased cell apoptosis rate from (1.14-/+0.79)% to (22.47-/+3.15)% occurred following pSilencer U6 2.1-Lon transfection of the cells. CONCLUSIONS: Lon gene can be significantly downregulated by introduction of siRNA in MCF7 cells to result in enhanced sensitivity of MCF7 cells to UV, cisplatin and heat stress.

Silk sericin-insulin bioconjugates: synthesis, characterization and biological activity.

When silk fiber derived from Bombyx mori was subjected to degumming treatments twice in water and subsequent degraded processing in slightly alkaline aqueous solution under high-temperature and high-pressure, the water-soluble silk sericin peptides (SS) with different molecular mass from 10 to 70 kDa were obtained. The sericin peptides could be conjugated covalently with insulin alone with cross-linking reagent glutaraldehyde. The physicochemical properties of the silk sericin-insulin (SS-Ins) conjugates were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The biological activities of SS-Ins bioconjugates were investigated in vitro and in vivo. The results in human serum in vitro indicated that the half-life of the synthesized SS-Ins derivatives was 2.3 and 2.7 times more than that of bovine serum albumin-insulin (BSA-Ins) conjugates and intact insulin, respectively. The pharmacological activity of SS-Ins bioconjugates lengthened to 21 h in mice in vivo, which was over 4 times longer than that of the native insulin. The immunogenicity of silk sericin and the antigenicity of SS-Ins derivatives were not observed in both rabbits and mice. The bioconjugation of insulin with silk sericin protein evidently improved both physicochemical and biological stability of the polypeptide.

Synthesis of silk fibroin-insulin bioconjugates and their characterization and activities in vivo.

The regenerated liquid silk fibroin with an average molecular mass of about 60 kDa consists of 18 kinds of amino acids containing approximately 10% of polar amino acids with hydroxyl and amino groups such as serine and lysine. The liquid silk fibroin is coupled covalently with insulin molecules through these strongly polar side groups by using glutaraldehyde. The physicochemical properties of the silk fibroin-insulin (SF-Ins) bioconjugates were investigated by enzyme-linked immunosorbent assay for the quantitative measurement of insulin. The biological activities of the insulin bioconjugates were characterized in vitro and in vivo. The SF-Ins constructs obtained by 5 h of covalent crosslinking showed much higher recovery (about 70%) and in vitro stability in human serum than bovine serum albumin-insulin (BSA-Ins) derivatives. The results in human serum indicated that the half-life in vitro of the biosynthesized SF-Ins derivatives was 2.1 and 1.7 times more than that of BSA-Ins conjugates and native insulin, respectively. The immunogenicity of the regenerated silk fibroin and the antigenicity of silk fibroin-modified insulin were not observed in both rabbits and rats. The pharmacological activity of the SF-Ins bioconjugates in diabetic rats evidently lengthened and was about 3.5 times as long as that of the native insulin, nearly 21 h. The bioconjugation of insulin with the regenerated silk fibroin greatly improved its physicochemical and biological stability.

[Apoptotic characteristics of spleen lymphocyte in mice irradiated by lethal dose and its relationship to the expression of Bax and Bcl-XL proteins].

OBJECTIVE: To observe the apoptotic characteristics of mouse spleen lymphocyte after lethal dose gamma-irradiation and its relationship to the expression of Bax and Bcl-XL proteins. METHODS: Two hundred and twenty-five second-grade C57 mice were randomly divided into six groups of 0, 6, 9, 12, 15 and 20 Gy. They were sacrificed by dislocation and samples were taken on 1-28 days after whole body single gamma-irradiation. Lymphocyte apoptosis and necrosis were analyzed by TdT-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM) techniques. The expression of Bax and Bcl-XL proteins were estimated by immunohistochemical method. RESULTS: (1) The number of peripheral white blood cells of mice increased temporarily at 6 hours after radiation, thereafter, began to decrease rapidly, which reached the minimum on day 7 and recovered normal level basically one month after 6 Gy gamma-irradiation. (2) Apoptotic rate of spleen lymphocytes increased significantly, peaking at 6 hours after radiation, which was found to have a dose-response relationship during 6-24 hours after < or =12 Gy irradiation, but decreased after > or =15 Gy irradiation. (3) It was confirmed by FCM that the apoptotic rate of spleen lymphocytes increased along with the elevation of radiation dose. However, the apoptotic rate began to decrease and the necrotic rate rose distinctively after > or =15 Gy irradiation. The analysis of DNA gel electrophoresis supported above-mentioned results. (4) The expression of Bax protein in spleen lymphocyte enhanced at 6 hours after 6 Gy gamma-irradiation and peaked by 12 Gy-irradiation, showing a dose-dependent pattern, but which was not be found after > or =15 Gy gamma-irradiation. On the other hand, the expression of Bcl-XL protein reduced persistently with the increase of radiation dose, and also presented a better dose-dependent effect after < or =12 Gy irradiation. CONCLUSION: After 6-12 Gy gamma-irradiation, the apoptosis is the major death way of spleen lymphocyte, while both necrosis and apoptosis are important death pathways after > or =15 Gy irradiation. Pro-apoptotic Bax and anti-apoptotic Bcl-XL play an important role in the apoptotic regulation of spleen lymphocytes induced by lethal dose radiation.

[The effects of acute large dose of gamma-irradiation on immune function of mice].

AIM: To observe the effects of large dose of gamma-irradiation on immune function of mice. METHODS: 225 cleaning-grad C57 mice, weighing(20+/-2.0) g, were randomly divided into 6 groups, and treated with 0, 6, 9, 12, 15 and 20 Gy gamma-irradiation. At different times after irradiation, lymphocytes were collected and lymphocytic apoptosis and T cell subsets were analyzed by TUNEL, May-Grunwald Giemsa (MGG) staining and flow cytometry. RESULTS: (1)At early stage(1-14) d after radiation, the apoptotic rate of peripheral blood lymphocytes increased significantly and 12 Gy radiation resulted in the highest apoptotic rate. The number of T lymphocytes and T cell subsets decreased continuously in a dose-dependent manner. CD8(+) T cells were the most sensitive in T cell subsets to irradiation. These results suggested that early severe injury might be one of the important features of immune injury caused by acute radiation. (2) One month after radiation, the apoptotic rate of lymphocytes began to decrease and T lymphocytes and their subsets recovered gradually. However, neither the lymphocytic apoptotic rate nor the number of CD3(+) T cells and CD8(+) T cells, recovered to normal level, indicating that large dose of radiation had severe remote effects on immune function. CONCLUSION: Apoptosis of a large number of peripheral blood lymphocytes in early stage after radiation may result in sharp reduction of T cell number and late immune function depression.

[Relationship between apoptosis of mouse thymic lymphocytes and expressions of bax, bcl-2 and bcl-XL after gamma-ray radiation with lethal dose].

AIM: To observe the relationship between apoptosis of mouse thymic lymphocytes and the expressions of bax, bcl-2 and bcl-X(L) after gamma-ray radiation with lethal dose and provide the basis for treatment of acute severe radiation sickness. METHODS: 250 cleaning-grade C57 mice were randomly divided into 6 groups and treated with 0, 6, 9, 12, 15 and 20 Gy of whole body single gamma-irradiation, respectivety. The mice were killed by dislocation and then the thymus and peripheral blood samples were taken at 1-28 days and 3-12 months after irradiation. The lymphocytic apoptosis was analyzed by TUNEL. The expressions of bax, bcl-2 and bcl-X(L) were detected by in situ hybridization and immunohistochemical staining. RESULTS: (1)Mouse peripheral leucocytes showed a transient elevation at 6 h after radiation and then decreased rapidly. The leucocyte's number reduced to minimal level at day 7 after 6 Gy gamma-irradiation and returned to basic normal value until one month after radiation. (2)24 h after irradiation the apoptotic rate of thymic lymphocytes increased swiftly, and apoptotic rate was positively correlated with radiation dose in the range of 6-12 Gy. There was no such a correlation after >/=15 Gy irradiation. (3)24 h after 6 Gy irradiation the apoptotic rate of thymic lymphocytes reached the maximal level. Afterwards it began to decrease, but still higher than that of control group even after 12 months. (4)3 h after 6 Gy radiation the expression of Bax protein in thymic lymphocytes increased immediately, and reached the highest value at 24 h. On the other hand, the expressions of Bcl-2 and Bcl-X(L) proteins reduced evidently at 3 h after radiation, reaching the lowest level at 24 h. Analysis of bax and bcl-2 mRNAs was concordant with the protein expression results. CONCLUSION: After 6-12 Gy gamma-ray irradiation, the apoptotic rate of thymic lymphocytes is positively correlated with the radiation doses, suggesting that apoptosis is a major way of thymic lymphocyte death after

[Effect of papillary thyroid carcinoma related gene PTC1 on subcellular localization and function of ATM cell].

BACKGROUND & OBJECTIVE: Papillary thyroid carcinoma is characterized by RET (rearranged during transfection)/PTC (papillary thyroid carcinoma) rearrangements. However, the function of RET/PTC in carcino- genesis is not well understood. This study was designed to investigate the interaction between DNA double-strand break sensor ATM (mutated in ataxia telangiectasia) kinase and PTC1, a rearranged form of proto-oncogene ret, to explore the role of ret rearrangements in carcinogenesis. METHODS: RET TK phosphorylation was determined by in vitro kinase assay using the immunoprecipitation with anti-ATM antibody as kinase and the immunoprecipitation of HA-tagged TK as substrate. The location of ATM-LZPR in COS7 cells coexpressed with PTC1 was investigated by protein extracts of cytoplasm and nucleus. The phosphorylation level of p53, was determined by Western blot analysis with the antibody against phosphorylated p53, and the cell cycle was determined by flow cytometry when PTC1 overexpressed. RESULTS: ATM directly phosphorylated TK domain of PTC1 in vitro kinase assay. ATM-LZPR was located in both cell cytoplasm and nucleus when PTC1 was not expressed, however, co-overexpression of PTC1 and ATM-LZPR made the latter locate only in the cytoplasm. In addition, overexpression of PTC1 inhibited the phosphorylation level of p53 by ATM and caused G(1)/S phase arrest of cell cycle. CONCLUSIONS: PTC1 may remain ATM kinase in cytoplasm and inhibit the phosphorylation of p53 by ATM. PTC1, a rearrangement form of ret, may result in the disorder of cell damage repair and cell cycle checkpoint and destroy cell homeostasis.