CN103919806A - Application of mesenchymal stem cells as medicine for treating normal hexane-induced peripheral neuropathy - Google Patents
Application of mesenchymal stem cells as medicine for treating normal hexane-induced peripheral neuropathy Download PDFInfo
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Abstract
本发明所述的骨髓间充质干细胞（mesenchymalstemcells，MSCs）的具体应用，解决了传统上没有针对正己烷致周围性神经病进行有效治疗的药物的问题，为治疗该种神经病提出了新的思路和具体应用，它以患有正己烷致周围性神经病的大鼠作为实验对象，通过步态评分，神经电生理（神经传导潜伏期、神经传导速度），脊髓和坐骨神经的砂罗铬花青染色、扫描电镜、髓鞘碱性蛋白和低分子量神经丝为检测指标；结果显示，MSCs作为一种药物，对正己烷致周围性神经病大鼠有显著的修复作用，它将在职业中毒致周围性神经病的治疗中发挥重要作用。
The specific application of bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) in the present invention solves the traditional problem that there is no effective drug for the treatment of n-hexane-induced peripheral neuropathy, and proposes new ideas and ideas for the treatment of such neuropathy. For specific applications, it takes rats with n-hexane-induced peripheral neuropathy as experimental subjects, through gait score, nerve electrophysiology (nerve conduction latency, nerve conduction velocity), saura chrome cyanine staining and scanning of spinal cord and sciatic nerve Electron microscope, myelin basic protein and low-molecular-weight neurofilament were the detection indicators; the results showed that MSCs, as a drug, had a significant repair effect on the peripheral neuropathy rats induced by n-hexane, and it would prevent the peripheral neuropathy caused by occupational poisoning. important role in treatment.
Description
Technical field
The present invention relates to a kind of application of mesenchymal stem cells MSCs, particularly a kind of mesenchymal stem cells MSCs causes the application of the neuropathic medicine of property around as treatment normal hexane.
Background technology
Chronic n-hexane poisoning patient's nervous system injury (normal hexane causes property neuropathy around) is that 5-acetyl butyryl (HD) causes by final metabolite and the whole poisonous substance 2 of normal hexane.Its pathological characteristic is that the neurofilament of axon gathers and segmental swelling, and the change of secondary segmental demyelination, the most obvious with length, crude fibre.Many scholars, from aspects such as axon skelemin, energy metabolism impairment and NGF signal transduction pathways, are studied the pathogenesis of poisonous peripheral neuropathy caused by N-hexane, but not yet very clear so far.Chronic n-hexane poisoning patient's treatment at present, comprise disengage, ensure that patient has enough nutrition, gives vitamin B group, the Chinese medicine of Energy mixture, blood circulation promoting and blood stasis dispelling, dredging collateral the kidney invigorating and be aided with acupuncture, physical therapy and quadruped locomotion functional exercise, there is no special effect medicine therapeutic.Therefore need to find or develop now one can treat normal hexane and cause the neuropathic medicine of property around.
Summary of the invention
The present invention is in order to solve the existing above-mentioned deficiency of prior art, proposes a kind ofly can treat the related application, particularly mesenchymal stem cells MSCs that normal hexane causes property neuropathy medicine around and cause the application of the neuropathic medicine of property around as treatment normal hexane.
Technical solution of the present invention is: a kind of mesenchymal stem cells MSCs causes the application of the neuropathic medicine of property around as treatment normal hexane.
Compared with the existing technology, tool has the following advantages in the present invention:
Mesenchymal stem cells MSCs of the present invention (mesenchymal stem cells, MSCs) concrete application, solve traditionally and carried out the effectively problem of the medicine for the treatment of less than cause property neuropathy around for normal hexane, for treating this kind of neuropathy, new thinking and concrete application are proposed, it causes around the neuropathic rat of property as experimental subject to suffer from normal hexane, mark by gait, Electrophysiology (nerve conduction incubation period, nerve conduction velocity), spinal cord and the dyeing of sciatic sand sieve eriochrome cyanine, scanning electron microscope, myelin basic protein (MBP) and low-molecular-weight neurofilament (low-molecular-weight neurofilament) are for detecting index, result shows, MSCs is as a kind of medicine, and normal hexane is caused to property neuropathy rat around significant repair, and it will be playing a significant role in the neuropathic treatment of property around professional poisoning induced.
Brief description of the drawings
The biological characteristics of Fig. 1 mesenchymal stem cells MSCs.
Fig. 2 MSCs transplants the impact on rat behavior ability.
Fig. 3 MSCs transplants the impact on rat Electrophysiology.
Sand sieve eriochrome cyanine dyeing (× 200) of Fig. 4 spinal cord transection face.
Cross section sand sieve of Fig. 5 sciatic nerve eriochrome cyanine dyeing (× 200).
The scanning electron microscope of Fig. 6 spinal cord transection face detects.
The cross section scanning electron microscope of Fig. 7 sciatic nerve detects.
Fig. 8 MSCs transplants the impact on myelin basic protein mrna expression.
Fig. 9 MSCs transplants the impact that myelin basic protein is expressed.
Figure 10 MSCs transplants the impact on low-molecular-weight neurofilament mrna expression.
Figure 11 MSCs transplants the impact that low-molecular-weight neural thread protein NTP is expressed.
Detailed description of the invention
First carry out the separation and Culture of mesenchymal stem cells MSCs (MSCs):
Rat dislocation is put to death, 75% ethanol submergence sterilization, aseptic femur and the tibia peeled off, remove metaphysis, medullary cavity is exposed, PBS rinses repeatedly, collecting cell is crossed screen cloth centrifugal, with containing the DMEM culture fluid re-suspended cell of 10% serum and being inoculated in culture dish, at 37 DEG C, CO
2in saturated humidity incubator, cultivate, after 24h, change liquid.3d changes liquid 1 time later, in the time that cell reaches 80% ~ 90% fusion, goes down to posterity, and utilizes progressively purification MSCs of differential attachment method.Get the good cell of the 3rd generation growth conditions, utilize monoclonal antibody CD90, CD45 to carry out flow cytometer detection, so that MSCs is identified.
Set up the rat model that normal hexane causes peripheral neuropathy:
Slight disease model---200 mg/kg 2,5-acetyl butyryl rats by intraperitoneal injection, 1 day 1 time, 1 week 5 times, continuous 6 weeks.
Severe disease model---400 mg/kg 2,5-acetyl butyryl rats by intraperitoneal injection, 1 day 1 time, 1 week 5 times, continuous 6 weeks
Set up the detection platform that normal hexane causes peripheral nerve disease model:
Ordinary circumstance is observed, and comprises developmental state, action, fur, diet, body weight etc.
Electrophysiology detects: rat etherization, ventricumbent position is positioned in rat fixator, exposes afterbody.Be labeled as respectively A, B, C point at afterbody apart from 3cm, 10cm, the 15cm place of afterbody starting point, A point is placed stimulating electrode, B point and C point are placed recording electrode, record between AB, nerve conduction incubation period between AC, be BC distance between two points according to formula MCV=Length BC/Latency BC(Length BC, Latency BC is BC point-to-point transmission latent time) calculating nerve conduction velocity.
Spinal cord and the dyeing of sciatic sand sieve eriochrome cyanine: paraffin section de-waxing is to water, according to test kit explanation, drip sand sieve eriochrome cyanine dyeing 20min, flowing water is washed 2min, drip ammonium ferric sulfate, be light gray to collagen fiber, myelin is clear blueness, and flowing water rinses 5min, drip phloxine and redye the several seconds, slightly washing, conventional dehydration is transparent, neutral gum mounting.
Spinal cord and sciatic surface sweeping Electronic Speculum detect: rat anesthesia, fixing, fixative is the phosphate buffer (pH=7.4) of 2.5% paraformaldehyde+2.0% glutaraldehyde 0.1mol/L, after fixedly completing, gets spinal cord and sciatic nerve.By spinal cord and sciatic nerve, be placed in above-mentioned infusion liquid and continue fixing 24h rapidly, 10g/L osmic acid is fixed 1h, the dehydration of acetone gradient, Epon812 embedding, ultrathin section, acetic acid uranium and lead citrate double staining, under 80kV voltage with H-600 type transmission electron microscope observing and take the photograph sheet.
Spinal cord and sciatic myelin basic protein detect: detect the mRNA level of myelin basic protein and the expression of protein level with real time PCR and Western blot respectively.
Spinal cord and sciatic low-molecular-weight neurofilament detect: detect the mRNA level of low-molecular-weight neurofilament and the expression of protein level with real time PCR and Western blot respectively.
MSCs transplants normal hexane and causes peripheral neuropathy rat:
Every rat model tail vein transplantation 1 × 10
6mSCs.Detect according to the operating procedure of setting up normal hexane and cause peripheral nerve disease model detection platform.
Result
Rat marrow mesenchymal stem cells MSCs In vitro culture and qualification:
After primary cell inoculation 72h, most cells is adherent, and after 10 ~ 15 days, cell reaches 80% ~ 90% fusion, goes down to posterity.After cultivating for 3 generations, cellular morphology trend homogeneous, similar fibroblast sample, is pencil or whirlpool shape ordered arrangement (Figure 1A).Get the 3rd generation cell carry out flow cytometer detection, the positive expression rate of surface molecular CD90 and CD45 be respectively 96.61% and 0.40%(Figure 1B, 1C).Cultivating gained cell is to transplant available MSCs.
In Figure 1A, third generation mesenchymal stem cells MSCs, cellular morphology homogeneous, similar fibroblast sample, is pencil or whirlpool shape ordered arrangement; In Figure 1B, flow cytometer detection shows 96.61% cellular expression CD90; In Fig. 1 C, flow cytometer detection shows 0.40% cellular expression CD45.
Normal hexane causes the foundation of peripheral nerve disease model:
That control animals is grown is normal, action rapidly, hair color is glossy, in experimentation without abnormal gait.Poisoning rat model is ingested, and minimizing, hair color are dark and gloomy, be slow in action even paralysis, body weight gain slowly even decline.To contaminating the 5th week, calomel poisoning rat occur significantly taking knee joint decline and hangover as the dyskinesia of feature, severe intoxication rat hindlimb draw, cannot be stood completely, rear palm turns over, gait is marked and is all increased gradually with the prolongation of contamination time.Control rats gait is normal all the time.
MSCs transplants the impact that normal hexane is caused to peripheral neuropathy:
Behavioral competence is observed: after MSCs transplants, the behavioral competence of slight and severe rat model all be improved significantly (Fig. 2).
Fig. 2 A, Fig. 2 D are normal control rat, and activity freely; Fig. 2 B is calomel poisoning rat, occurs significantly declining and dragging the dyskinesia of Fig. 2 A tail as feature taking knee joint; After Fig. 2 C is calomel poisoning rat implantation MSCs, hind leg abduction degree alleviates; Fig. 2 E is severe intoxication rat, occurs that hind leg is dilatory and cannot stand completely, and rear palm turns over; After Fig. 2 F is severe intoxication rat implantation MSCs, slightly recover, rear palm turns over disappearance, and hind leg can support ground, but has abduction.
Electrophysiology detects: after MSCs transplants, nerve conduction slight, severe rat model shortens incubation period gradually, nerve conduction velocity accelerate gradually (Fig. 3 A, 3B).MSCs transplants the Electrophysiology that can obviously improve normal hexane and cause peripheral neuropathy rat.
Can find out by Fig. 3 A, after MSCs transplants, the nerve conduction velocity of poisoning rat is accelerated gradually, basic recovery normally; Can find out by Fig. 3 B, after MSCs transplants, the nerve conduction of poisoning rat shortens incubation period gradually, basic approaching normally.
Spinal cord and sciatic sand sieve eriochrome cyanine dyeing: obvious demyelination and nerve fiber arrangement disorder all appear in the spinal cord of poisoning rat and sciatic nerve, after MSCs transplants demyelination and nerve fiber arrangement disorder phenomenon all be improved significantly (Fig. 4, Fig. 5).
Fig. 4 A, spinal cord of normal white matter compact structure is regular; Fig. 4 B, calomel poisoning rat, substantia alba medullae spinalis short texture, there is demyelination in subregion; Fig. 4 C, calomel poisoning rat implantation MSCs, substantia alba medullae spinalis structure is finer and close, and demyelination phenomenon part is recovered; Fig. 4 D, severe intoxication rat, serious demyelination, subregion is free bubble structure to form; Fig. 4 E, severe intoxication rat implantation MSCs, demyelination phenomenon is improved, and cavity structure disappears substantially.
Fig. 5 A, normal rat, myelinated fiber is densely distributed and even, and single fiber is full; Fig. 5 B, calomel poisoning rat, axon is sparse, and there is atrophy in part aixs cylinder; Fig. 5 C, calomel poisoning rat implantation MSCs, axonal atrophy phenomenon part is improved; Fig. 5 D, severe intoxication rat, the sparse arrangement of axon, there is obvious atrophy in aixs cylinder; Fig. 5 E, severe intoxication rat implantation MSCs, axon gap obviously dwindles, and axonal atrophy is obviously improved.
Spinal cord and sciatic scanning electron microscope detect: after MSCs transplants, the spinal cord demyelination of poisoning rat and spinal cord layering increase phenomenon and obviously improve, and sciatic myelin caves in and neurofilament Density inhomogeneity phenomenon disappears substantially (Fig. 6, Fig. 7).
Fig. 6 A. normal rat, myelin structural integrity; Fig. 6 B, calomel poisoning rat, part myelin caves in to aixs cylinder, occurs slight demyelination; Fig. 6 C, calomel poisoning rat implantation MSCs, myelin female recovers, and demyelination phenomenon part disappears; Fig. 6 D, severe intoxication rat, serious demyelination, myelin layering showed increased; Fig. 6 E, severe intoxication rat implantation MSCs, demyelination phenomenon part is recovered, and myelin layered portion reduces.
Fig. 7 A, normal rat, myelin structural integrity; Fig. 7 B, calomel poisoning rat, part myelin caves in to aixs cylinder, atrophy that aixs cylinder is slight; Fig. 7 C, calomel poisoning rat implantation MSCs, myelin female recovers, and it is normal that aixs cylinder part is recovered; Fig. 7 D, severe intoxication rat, myelin depression, the obvious atrophy of aixs cylinder; Fig. 7 E, severe intoxication rat implantation MSCs, myelin female recovers, and it is normal that aixs cylinder part is recovered.
Spinal cord and sciatic myelin basic protein detect: P
0be a kind of major structural protein on peripheral nerve myelin sheath, account for the more than 50% of histone total amount, in neural myelin tissue, brought into play the important function such as adhesion, signal transduction around.Real time PCR and WB detect all and show, the mrna expression level of myelin basic protein and all significantly recoveries (Fig. 8, Fig. 9) of protein expression level in poisoning rat spinal cord and sciatic nerve after MSCs transplants.
Fig. 8 A and Fig. 8 B, MSCs transplants impact slight, severe intoxication rat spinal cord myelin basic protein mrna expression; Fig. 8 C and Fig. 8 D, MSCs transplants impact slight, severe intoxication rat sciatic nerve myelin basic protein mrna expression.
Spinal cord and sciatic low-molecular-weight neurofilament (low-molecular-weight neurofilament) detect: NF maintaining form and the motor function of neurocyte, maintain in the steric configuration of aixs cylinder and normal Neurotransmission speed thereof and play an important role.2,5-HD contamination has disturbed NF to assemble normally and go the balance between assembling, has disturbed the dynamic exchange between the NF mesh skeleton in NF monomer and the static pond in dynamic pond, therefore cause neurofilament structure disturbance.After MSCs transplants, low-molecular-weight neurofilament level is obviously recovered (Figure 10, Figure 11).
Figure 10 A and Figure 10 B, MSCs transplants impact slight, severe intoxication rat spinal cord low-molecular-weight neurofilament mrna expression; Figure 10 C and Figure 10 D, MSCs transplants the impact on poisoning rat sciatic nerve low-molecular-weight neurofilament mrna expression.
Figure 11 A and Figure 11 B, MSCs transplants the impact that poisoning rat spinal cord low-molecular-weight neural thread protein NTP is expressed; Figure 11 C, and Figure 11 D, MSCs transplants the impact that poisoning rat sciatic nerve low-molecular-weight neural thread protein NTP is expressed.
In sum, MSCs is transplanted to normal hexane and causes around in property neuropathy rat body, can significantly repair the organizational structure of its damaged nerve tissue, improves Electrophysiology function, and makes its neurobehavioral trend normal.MSCs can be used as treatment normal hexane and causes the neuropathic a kind of new method of property around.
Claims (1)
1. a mesenchymal stem cells MSCs causes the application of the neuropathic medicine of property around as treatment normal hexane.
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