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New Year's Eve

Today,it is the Chinese traditionary festival:Spring Festival.

My Experimental Research for Doctor Degree:Part-3,4

Part 3 Expression of the recombinant plasmid of pDsVEGF165Red1-N1 or pIRES2- BMP2- EGFP in mammalian cells

Objective To detect the efficiency of the recombinant plasmids, pDsVEGF165 Red1-N1 and pIRES2-BMP2-EGFP, in mammalian cells and to provide the foundation of gene therapy in vivo. Methods 293-T cells grown in six-well plates at a density of 1×105 cells in 0.5 ml of DMEM per well were transfected with pDsVEGF165Red1-N1 and pIRES2-BMP2-EGFP plasmids DNA or pDsRed1-N1 and pIRES2-EGFP empty vector DNA by means of DOTAP in condition recommended by the manufacture（Roche）.Plasmid DNA (total 2.5 ug of two plasmids) was dissolved in 25 ml HBS and mixed with 15 ml DOTAP reagent dissolved in 50 ml HBS, and incubated at 15℃ for 15 min to allow DNA-DOTAP complexs to form. Then 0.5 ml medium without serum was added, and the DNA-DOTAP solution was applied onto a monolayer of 1×105 cells in six-well plates. The cells were incubated with the complexes for 6 h at 37℃ 5％CO2 incubator. Following incubation , 1 ml of growth medium containing 10% serum was added and removing the transfection mixture. 48 h later,DsRed and EGFP were observed by fluorescent microscope and confocal laser-scanning microscope,mRNA of genes were detected by RT-PCR and proteins expressed in cells were detected by Western Blotting. Results Recombinant plasmids pDsVEGF165Red1-N1 and pIRES2-BMP2- EGFP all allow proteins transient expression in mammalian cells, which was confirmed by fluorescent microscope,CLSM and Western Blotting. Conclusion Transfection of the mammalian cells with plasmids pDsVEGF165 Red1-N1 and pIRES2-BMP2-EGFP leads to transient expression VEGF165 or BMP2.

If you want to read the picture about the part 4, please see the link:

http://haibo-spine.blog.sohu.com/109142477.html

Part 4 The function of recombinant plasmids pDsVEGF165Red1-N1、pIRES2- BMP2- EGFP in long bone defect of rabbits

Objective To discuss the function of recombinant plasmid, pDsVEGF 165Red1-N1、pIRES2-BMP2-EGFP, in neovascularication and bone formation of long bone defect of rabbits. Methods A total of 30 healthy adult rabbits were randomly divided into five groups. Right forelimb was operated as experimental limb.The first group: collagen membrane,n-HA/PA, pDsVEGF165Red1-N1 and pIRES2-BMP2-EGFP plasmids were applied; the second group: collagen membrane,n-HA/PA, pDsVEGF165Red1- N1 plasmids were applied; the third group: collagen membrane, n-HA/PA,pIRES2-BMP2- EGFP plasmids were applied; the fourth group: collagen membrane, n- HA/PA (no combined plasmid DNA) were applied; the fifth group: Constructing the long bone defect, no treatment. The samples were examined by gross, X-ray, histomorphology, SEM and ECT in 2W, 4W,8W. Results (1) The expression of fluorescent (EGFP or RFP) can be observed by fluorescent microscope in muscles around the bone defect at two, four, eight weeks; (2) At 2 weeks, no distinct difference among groups was observed. ECT showed that the blood flow of the first group in the bone defect site was increased(P<0.05)、、、、、、>

If you want to read the picture about the part 4, please see the link:

http://haibo-spine.blog.sohu.com/109142708.html

My Experimental Research for Doctor Degree:Abstract,Part-1,2

THE PRIMARY RESEARCH ON REPAIR OF LONG BONE DEFECT USING CONSTRUCTED pDsVEGF165Red1-N1, pIRES2-BMP2-EGFP EUKARYOTIC EXPRESSIVE PLASMIDS

ABSTRACT
With the deep study of long bone defect, its treatment is from traditional methods,such as autologous bone,allograft,to bone tissue engineering and gene engineering. To date, these tissue engineered construct systems are mainly applied to animal models not to human transplantation. A critical limiting factor in many of these investigations may be the lack of a functional vascular network in the engineered tissues.Thus, it has become a critical problem how to accelerate the revascularization in the process of repair of long bone defect. Recent evidence suggests that VEGF is considered to be a key mediator in the angiogenic process required during bone repair process and remodeling. BMPs can stimulate angiogenesis through the production of VEGF by osteoblasts. Meantimes, VEGF and BMPs act synergistically to enchance both bone formation and bone healing. The main purpose of the present study is the revascularzation of long bone defect through transfecting VEGF165 and BMP2 eukaryotic expressive plasmids in vivo, which was adehered to n-HA/PA66 at first. As the same time, we used the technique of guided bone regeneration (GBR) which was guided by BME-10X collagen membrane. There are four separate parts in this study.

If you want to read the picture about abstract, please see the link:

http://haibo-spine.blog.sohu.com/109141072.html

Part 1 Construction and identification of red fluorescent protein reporter gene vector containing human vascular endothelial growth factor 165

Objective :To construct the vector that expresses the fusion protein of VEGF165 and red fluorescent protein (RFP) in mammalian cells. Methods According to the nucleotide sequence of hVEGF165, a pair of oligonucleotides was designed as primers which contained nuncleotide sequence of HindⅢ and SacⅡ restriction endonuclease at the two end respectively. The sequence encoding for hVEGF165 （573bp）without stop codn was amplified using PCR technique. The PCR product was digested with HindⅢ and SacⅡ, and cloned into the pDsRed1-N1 plasmid containing the reporter gene RFP. The recombinant plasmid pDsVEGF165Red-N1 was identified by restriction endonuclease enzyme analysis and DNA sequence analysis. Results The recombinant fusion protein expression vector was verified correctly by enzyme digestion, PCR and sequence analysis. Conclusion A red fluorescent protein reporter gene vector containing human vascular endothelial growth factor sequence has been constructed successfully.Thus providing an important and convenient tool to study intracellular localization of VEGF and therapy treatment in vivo.

If you want to read the picture about the part 4, please see the link:

http://haibo-spine.blog.sohu.com/109141791.html

Part 2 Construction and identification of the pIRES2-Enchanced Green fluorescent protein reporter gene vector carrying BMP2 Gene

Objective To construct the pIRES2-enchanced green flurescent protein(EGFP) expression vector carrying BMP2 gene in order to provide an ideal reporter gene for the expression and identify the location of portein in Vitro and Vivo. Methods The BMP2 cDNA sequence was excised using EcoR I and Xba I, and subcloned on pUC18 vector.And then the BMP2 sequence on the pUC18 plasmid was excised with Sal I again and ligated into Sal I site of pIRES2-EGFP treated by phosphorylase in sense and antisense direction.First, positive clones were selected with Sal I excising the recombinant plasmid;then the insert direction of BMP2 was proved using EcoR I、PshA I. Last the DNA sequenceing was used for the analysis of BMP2 sequence. Results The recombinant expression vector was verified correctly by enzyme digestion, sequence analysis. Conclusion A pIRES2 Green fluorescent protein reporter gene vector containing human Bone Morphogenetic Protein2 has been constructed successfully. thus providing an important and convenient tool to study intracellular localization of BMP2.

If you want to read the picture about the part 4, please see the link:

http://haibo-spine.blog.sohu.com/109141868.html

How to exercise your back muscles.

Now ,I am watching the CNN :Obama steps into history as first black president

President George W. Bush speaks as President-elect Barack Obama looks over his shoulder during a meeting with former Presidents in the Oval Office, January 7, 2009.

How to do physical exercise for preventing osteoporosis?

Regular physical activity on a long-term basis has a particularly important role in maintaining healthy bones. Exercise can maintain and increase bone strength by increasing bone mass or by slowing age-related bone loss. Muscle strength is also increased, which is important for supporting the joints and preventing falls.
Exercise has also been shown to improve co-ordination and balance, which helps to prevent falls and to improve general physical health and well-being.
Be aware that any positive gains in bone strength are lost when you stop exercising, so that it is important that your exercise is regular and ongoing.
Caution: Someone who has established osteoporosis (one or more fractures) may not be able to do as many types of activities as someone without osteoporosis. Talk to your doctor and physiotherapist about activities you can do.
Exercise helps to build and maintain strong bones, prevent falls and fractures and speed rehabilitation.
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Physical Activity at Different Ages and Stages

Childhood /Adolescence
Bone status:In girls, the major build up of bone occurs in the pre-teen years. Lifetime Peak Bone Density is reached during mid to late 20s.Effect of exercise at this age:Maximises peak bone density, which helps keep bones strong for longer in adulthood.
Early to Middle Adulthood
Bone status:Bone loss starts to occur very gradually when a person reaches their thirties, although increases in bone density are still possible during middle adulthood. In women from 45 years on, bone loss begins to increase to 1- 2% per year.Effect of exercise at this age:Maintains bone strength by helping to slow bone loss, plus improve muscle strength, heart and lung fitness.
Postmenopausal Women
Bone status:
Bone loss speeds up to 2-4% per year at the onset of menopause.Effect of exercise at this age:Can maintain bone strength by helping to slow the rate of bone loss following menopause.
Men
Bone status:Generally bone density tends to remain constant until later in life. Low testosterone or hypogonadism can cause bone loss similar to postmenopausal women.Effect of exercise at this age:Improvements in muscular strength, balance and coordination to help prevent falls and maintain general health. Testosterone combined with exercise may be beneficial for bone in men with hypogonadism.
Older Adults without osteoporosis
Bone status:After 75 years of age further increases in bone loss occur in both sexes, especially from the neck of the femur (thigh bone). The risk of fracture increases as bone loss increases.Effect of exercise at this age:Helps to maintain bone strength and increase muscle strength, balance and co-ordination, which in turn help to prevent falls.
Older Adults with Osteoporosis/Fractures:
Bone status:Bones are increasingly thinner and brittle.Effect of exercise at this age:Exercises recommended by physiotherapists can improve general health, strength, balance and posture to prevent falls.
Exercises for bone health:
(assumes you don't have osteoporosis, otherwise see section below on exercise if you have osteoporosis)
There are two main types of exercises that are beneficial to bone health:
Weight-bearing exercise Resistance exercise (lifting weights with arms or legs)
Weight bearing exercise
Weight-bearing exercise means any exercise that is done while you are on your feet, so that gravity is exerting a force.
Your bones become stronger when they bear weight during exercise and when some amount of 'impact' or extra strain is placed on those bones.
If the 'strain' level is too low, then the bone will not become stronger and may still lose mass. Too much 'strain' could result in injury. The best 'strain' is from activities that may be new to your body, which means your bones are getting a variety of forces and loads on them.

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Exercise: if you have osteoporosis
If you have been diagnosed with osteoporosis and/or have already had a fracture, you should see your doctor or physiotherapist about starting an individual exercise program.
Your aim is to maintain bone strength and reduce the risk of fractures and falls by improving posture, balance and muscular strength.
Because of the higher risk of fracture, exercising at a level high enough to produce stress on the bones is not recommended.
Appropriate exercises include Tai Chi for balance, flexibility and muscle strength, gentle weights for muscle strength, water-based exercises (hydrotherapy) and walking.
Avoid high impact activities (eg running).
Avoid jarring & twisting movements (eg golf swing).
Avoid heavy lifting and lifting objects some distance from your body (eg picking up grandchildren).
Avoid abdominal curls (sit-ups).
Avoid bending forward from the waist.
Avoid sudden, abrupt movements.
Don't overdo it (especially if you have not done any activity for many years).

The Situation of Osteoporosis in China

Now, I am working at the orthopaedic outpatient.This morning, I saw many osteoporosis patients and there were two famale patiens having fracture of vertebral.They should be operated using PVP(Percutaneous VertebroPlasty).
In China,the science education about senile disease for example,OA,OP, and so on are very poor.So,I think when I have some idle time,I will take some lectures about the science education for the old persons.

The next websites are about osteoporosis in another countries.

http://www.mayoclinic.com/health/osteoporosis/DS00128

http://www.osteoporosis.ca/

http://www.osteoporosis.org.au/

http://orthoinfo.aaos.org/topic.cfm?topic=a00232

I buy one book:Dreams from My Father: A Story of Race and Inheritance

Today, I bought one book:Dreams from My Father: A Story of Race and Inheritance .It is China Edition.

About Twin Cities Spine Center

Recently,I read the websits:
http://www.tcspine.com/

On internet,there is some introduction about the hospital:
https://healthpartners.com/locator/practitioner/detail.do?personnelId=4506

http://twincities.citysearch.com/profile/5579792/minneapolis_mn/twin_cities_spine_center.html

Today, I creat a sohu blog.

One friend advise me to creat a blog at SOHU(WWW.SOHU.COM) website.

http://haibo-spine.blog.sohu.com/

Now,I am working in the orthopaedic out-patient

This morning ,I am visiting patients at the orthopaedic outpatient.
There are many patients everyday.Most patients are OA,OP or LDH.
Some patients are very confused how to treat these diseases.
In China,the science education is poor.